Biochemical Engineering Journal 113 (2016) 6676

Contents lists available at ScienceDirect

Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Quantitative evaluation of the shear threshold on Carthamus tinctorius

L. cell growth with computational uid dynamics in shaken ask
bioreactors
Yu Liu a , Ze-Jian Wang a,b, , JianWen Zhang a , Jian-ye Xia a , Ju Chu a , Si-Liang Zhang a ,
Ying-Ping Zhuang a
a
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai Institute of Biomanufacturing Technology &
Collaborative Innovation Center, Shanghai 200237, China
b
Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Quantitative evaluation of the shear threshold on C. tinctorius L. cell growth is vital for bioreactor system
Received 18 December 2015 design and optimization of scaled-up industrial cultivation. The present research focused on investigating
Received in revised form 20 April 2016 the effects of shear force on C. tinctorius L. cell growth by computational uid dynamic (CFD) analysis
Accepted 2 June 2016
in shaken asks. The results revealed that specic cell growth rates were greatly inhibited as the shear
Available online 3 June 2016
force increased from 1.17 to 2.42 Pa. Recovery of viability and aggregation diameter to their normal
levels could be implemented after a 4-day adaptation with large uctuations in physiological state. With
Keywords:
further correlation analysis on shear force and C. tinctorius L. growth rate, a threshold value was identied
Fluid mechanics
Power consumption
at an average and maximum shear stress of approximately 0.55 Pa (0.06 w/kg) and 4.00 Pa (0.93 w/kg)
Aggregation according to the inuence on cell growth. Quantitative data on shear effects can facilitate the design of
Plant cell bioreactors industrial processes and lead to more rational scale-up in industrial C. tinctorius L. cultivation.
C. tinctorius L. cell 2016 Elsevier B.V. All rights reserved.
Stress thresholds

1. Introduction
Plant cell suspension cultivation has proven to be an effec-
tive method of producing many valuable bioproducts continuously
and stably with independence from geographical or environmental
variations and constraints [1]. C. tinctorius L. is one of the Chinese
herbal medicines most commonly used to prevent and treat cardiac
disease in clinical practice [2]. With the quickly increasing mar-
Abbreviations: a, specic gas-liquid interfacial area (m2 ); c*, saturated dissolved
oxygen (mmol/m3 ); co , concentration of oxygen in liquid (mmol/m3 ); d, largest inner
diameter of shake ask (mm); d0 , shaking diameter (mm); kL a, mass transfer coef-
cient (h1 ); n, shaking frequency or rotation speed (rpm); DL , diffusion coefcient
of oxygen (cm2/s); OTR, oxygen transfer rate (mmol kg1 h1 ); OUR, oxygen uptake
rate (mmol kg1 h1 ); Re, ask Reynolds number (); V, shake ask nominal vol-
ume (ml); Vl , lling volume of shake ask (ml); , turbulent energy dissipation
rate (m2 s3 or w/kg); , specic growth rate (h1 ); , uid density (kg m3 ); , tively evaluate the shear threshold on C. tinctorius L. cell growth,
dynamic uid viscosity(Pa s); t , shear stress from uctuating velocity gradient (Pa);
d , shear stress acting on opposite sides of the cell (Pa); , angular velocity (rad/s);
k , kolmogorov length scale (m).
Corresponding author at: State Key Laboratory of Bioreactor Engineering, East
China University of Science & Technology P.O. box 329, 130 Meilong Road, Shanghai
200237 Peoples Republic of China.
E-mail address: wangzejian@ecust.edu.cn (Z.-J. Wang).
ket demand, suspension callus cultures have become one of the
most commonly applied methods for C. tinctorius L.production.
Like many other plant cell suspensions cultivated for active sec-
ondary metabolites, a separate two-stage culture strategy has been
implemented for an industrial C. tinctorius L. cell culture process
[3]. In the late stage, elicitors are added to induce biosynthesis of the
secondary metabolites (Hydroxysafor yellow A and kaempferide)
[4]. To obtain the highest metabolite productivity, the most impor-
tant and difcult problem encountered is to obtain high cell density
and higher specic cell growth rates. However, in industrial C. tinc-
torius L. cell cultivation and scale-up in stirred tank bioreactors,
fragile and slow-growing effects have always been a serious prob-
lem. Hydrodynamic shear stresses generated by the turbulent ow
are thought to be the important inhibitory factor on growth [59].
However, hardly any reports could be found on C. tinctorius L. cell
shear tolerance. Therefore, it is of great importance to quantita-
which would be effectively applied for directing bioreactor system
design and scale-up optimization for industrial C. tinctorius L. cells.
Previous studies into the shear sensitivity of plant cells have
been carried out in well-dened shear devices, for example, couette
type viscometers, capillary devices, and submerged jet apparatuses
[1013]. The denable shear devices had serious deciencies such

http://dx.doi.org/10.1016/j.bej.2016.06.001
1369-703X/ 2016 Elsevier B.V. All rights reserved.
 Y. Liu et al. / Biochemical Engineering Journal 113 (2016) 6676
as unsuitability for long-term cultivation and insufcient oxygen
supply under low shear environments [14]. Furthermore, those
studies mainly focused on short duration exposure effects with
direct lethal or sub-lethal responses being considered [1013]. In
addition to the duration of exposure frequency, ow type (lami-
nar or turbulent) also needs to be taken into consideration [15,16].
n the other hand, in commonly used bioreactors, most of the
studies have concentrated on empirical correlations based on the
use of the parameters of rotation speed or impeller tip speed to
assess the effects of hydrodynamic stress [1720]. Obviously, these
parameters are always scale-dependent and difcult to evaluate
quantitatively.
Compared with other culture systems, shaken asks have
unique advantages in shear sensitivity studies: (i) suitability for
long-term cultivation; (ii) higher oxygen transfer rates under a low
shear environment; (iii) the distribution of shear force is relatively
uniform, with values of max /ave under 20; and (iv) it is possible to
quantify hydrodynamic stress by CFD techniques, which have been
widely used for shear environment investigations [2123].
The threshold tolerance of shear force varies greatly by cell line,
age, ow type and the frequency of exposure time [5]. The corre-
lation of various lethal and sub-lethal biological activities of carrot
cells with total energy dissipation was published by Dunlop et al.
[12]. Loss of mitochondrial activity was found at energy dissipation
higher than 1 103 w/kg. Furthermore, Takedas studies indicated
that ATP and NAD(P)H were reduced, while cytosolic calcium con-
tent increased at the level of 0.52.3 w/kg of energy dissipation
rate in C. tinctorius L. cell culture [24,25]. Moreover, in Siecks
recent study with animal cells, the threshold value of the average
energy dissipation rate is 0.4 w/kg, and higher levels would cause
productivity loss and a transcriptomic stress response. In addition,
a necrosis effect was discovered by Gregoriades and Tanzeglocks
studies, observing an energy dissipation rate from 1.0 to 4.0 w/kg
[26,27]. Similar results with a range from 0.59 to 1.19 Pa have been
reported for CHO cells, while for HEK cells, it is between 1.19 and
1.67 Pa (under laminar and turbulent conditions) [2832].
In this study, hydrodynamic stress was systematically analyzed
with CFD technology in commonly used shaken ask bioreactors
under various lled volume and rotation speed conditions. In com-
parison with only using energy dissipation rates to evaluate shear
damage, a more extensive characterization of turbulence through
eddy scale calculations was applied to determine the effect of
shear damage, which may be better for evaluating the effect on
large diameter plant aggregations. The sub-lethal effects caused by
hydrodynamics on C. tinctorius L. cells in a fed-batch process were
effectively evaluated. To develop an industrial plant cell bioreactor
design and the maximum operating range of hydrodynamic stress,
threshold values of hydrodynamic stress were determined.
2. Materials and methods
2.1. CFD model approach
Fluid ow was characterized by CFD with the commercial soft-
ware ANSYS CFX (CFX 11.0). The simulations were performed as a
two-phase ow using the volume of uid method. The VOF model
is a classical model that has been applied in many studies to sim-
ulate the movement of shaken bioreactors, and it has proved to
be an effective method of ow evaluation in shake asks [33]. The
working liquid was modeled as a Newtonian uid with the nominal
properties of water (e.g., density, viscosity, surface tension) and air
for the dispersed phase. The RNG k- turbulence model was used to
describe the turbulent ow because of signicant amounts of swirl
in the movement of the shaken ask [34].
67
In this research, we paid more attention to details of the calcu-
lation process such as mesh independent solution and Free Surface
model settings. Several renement steps were used to obtain a
grid-independent solution resulting in a nal mesh size of 1.1 106
computational elements. Meanwhile, three interface compression
levels were tested to mimic the air-water interface more real-
istically, and we use three different Volume Fraction Smoothing
Types to obtain a gradient of a smoothed volume fraction eld.
These details on the CFD models are shown in the Supplemen-
tary Information. The solver ran over at least 5 s (approximately
10 cycles at 115 rpm), and average turbulence energy dissipation
rates were monitored to ensure that the simulation would reach a
quasi-steady state (Supplementary material).
The ask movement was modeled following a previous works
centrifugal force drive method [23]. Two forces that drive the move-
ment of uid in the asks, gravity and centrifugal force, are formed
as the asks revolve. The centrifugal force is then translated into
movement of the uid in the ask with the formation of free surface.
The cyclic centrifugal force equation is as follows:
Fy = 2 r cos(t) (1)
Fy = 2 r sin(t) (2)
where is the angular velocity (rad/s), r is the shaking diameter
(50 mm), and t is the run duration (s).
2.2. Oxygen mass transfer modeling
Oxygen transfer coefcient always used for evaluating the ef-
ciency of bioreactors is used as one of the scale-up factors. The
interfacial mass transfer coefcient between the gas and the liquid
phase is expressed in terms of the interfacial area, a, and transfer
velocity, kl . In a shaken ask, a can be calculated with Eq. (3):
A
a= (3)
V
where A is the gas-liquid interface area (m2 ) and V is the liquid
volume (m3 ). The transfer velocity kl is estimated using the eddy
cell model proposed by Lamont and Scott [35] as follows (Eq. (4)):
  1/4
kL = K DL (4)

where K = 0.4 is the model constant. DL represents the diffusion
coefcient of oxygen at 25 C, and  is the liquid kinematic viscosity.
As oxygen mass transfer occurs at the interface, the local aver-
aged energy dissipation shows better agreement with reported
experimental data than volumetric averaged energy dissipation. So
here, is the face average energy dissipation rate at the gas-liquid
interface.
2.3. Shear force analysis
Under these conditions, the stress to which cells were exposed
was estimated using an approach previously developed by Soos
et al. and Tanzeglock et al. [27,36]. Briey, stress in the ow eld
depends on cell size relative to the turbulent eddy length scale (Kol-
 1/4
mogorov length scale) k = v3 / , where is the local turbulent
energy dissipation rate and  represents the kinematic viscosity.
If cell size is smaller than this length scale (dcell < k ) , any cell
damage is controlled by the local hydrodynamics within an eddy.
This type of shear force is described as t (for more details, see
Tanzeglock et al. [27,36]):
 
5
t =   (5)
2 6v v
where is the dynamic viscosity of the liquid.
68 Y. Liu et al. / Biochemical Engineering Journal 113 (2016) 6676
Fig. 1. Geometry and morphologies of the free surface (gasliquid interface) formed in unbafed and bafed asks generated by CFD.
A different situation arises when k is smaller than the cell size
or the cell resides in the inertial subrange (dcell > k ). The hydrody-
namic stress to which the cell is exposed is a result of the difference
between velocity uctuations at two points separated by a distance
D and is equal to [37]:
1 To estimate the operating range of hydrodynamic stress, pre-
d = C2 (D)2/3
2
with constant C2 approximately equal to 2 [38]. Assuming that
the separation distance, D, is comparable to the cell size dcell , we
nd:
2/3
d = (dcell )
In the present work, the diameter of C. tinctorius L. cell
aggregates typically ranges from 0.1 to 1.1 mm, while the Kol-
mogorov length is between 0.01 (EDR 102 w/kg) and 0.3 mm (EDR
104 w/kg). Animal cells (69 m) and microorganisms are small
compared with the Kolmogorov length scale k , d is small, and
t will be dominant, and because the energy dissipation rate has a
positive correlation with t , the energy dissipation rate can be used
as a characteristic value to evaluate the shear effect. However, plant
cells always grow in aggregates of from 2 to 1000 cells (0.05 mm
to well over 2 mm in diameter), so just evaluating shear damage
from the energy dissipation rate will fail to consider the effect of
different diameters.
2.4. Shaken asks used
Two types of asks, with bafes (characterized by conical bafe
depths of 15 mm to the bottom) and without (volume of 500 ml,
the maximum inner diameter d is 110 mm for all), were used in
the experiment (Fig. 1). The lling volume designated V was from
20% to 50% of the nominal volume, the shaking frequency was set
at 115220 rpm, and the shaking diameter d0 was 50 mm.
2.5. Cell suspension culture
C. tinctorius L. were maintained in Murashige and Skoog (MS)
medium supplemented with sucrose (30 g/L), NAA (102 mM), and
6-BA (103 mM). The pH was adjusted to 6.2 prior to sterilization.
Cell suspension cultures were conducted in 1000-mL Erlenmeyer
asks and subcultured every 7 days. Samples were taken daily
for growth and ofine data analysis. Foaming was controlled with
silicone-based antifoam at high shaking speed (concentration of
0.01% v/v).
(6) liminary experiments were conducted in four low hydrodynamic
environments (<0.41 Pa) in un-bafed shaken asks. In addition,
cultivation experiments were applied under ve high hydrody-
namic stress levels, ranging from 1.17 to 2.42 Pa, to evaluate the
effect of shear force on the plant cell cultures.
(7)
2.6. Off-line data measurements
Cell density and the integrity of the membrane were detected by
dry cell weight and capacitance measurement. The capacitance and
conductivity were measured with a capacitance electrode (Biomass
Monitor 220, Hamilton), and the performed frequency was 0.4 MHz
with polarization correction. During the capacitance measurement,
the probe remained covered in uid that was suspended with
cells but no shaking. The feasibility and stability of on-line viable
biomass detection in plant cell culture have been widely conrmed
by many researchers [39,40]. Preliminary work has been carried out
to assess the correlations between dry weight, fresh weight and
capacitance (Supplementary information) within the cell line. For
each experiment, the capacitance was maintained within the linear
range 0120 pF/cm to ensure the accuracy of the measurement.
A conductivity parameter was used to determine the con-
sumption of the major salt component in the medium (details in
Supplementary information), which is the main contributor to con-
ductivity (98.14%). Glucose, fructose, and sucrose were analyzed
using HPLC.
The viability of the cells was established according to the 2,3,5-
triphenyl-tetrazolium chloride (TTC) reduction method, which
correlates with mitochondrial activity [41].
A laboratory-scale focused beam reectance FBRM (model
G400) coupled with iC FBRM software from Mettler Toledo was
implemented to detect the particle size distribution in daily mea-
surements.
 Y. Liu et al. / Biochemical Engineering Journal 113 (2016) 6676
Table 1
Empirical formulas for kL a in the literature.
No. Reference
1 Henzler and Schedel [42]
2 Veljkovic et al. [43]
3 Nikakhtari & A. Hill [44]
4 Maier et al. [45]
5 C. Mrotzek et al. [46]
Fig. 2. Comparison of volumetric mass transfer coefcients (kl a) from the literature and CFD result as a function of the shaking frequency (n).
3. Results and discussion
3.1. CFD verication
Validation of the model was conducted by comparing simulated
gas-liquid mass transfer coefcient (kL a) results with others calcu-
lated in the literature by empirical formulas (Table 1). As shown
in Fig. 2, it could be clearly observed that the CFD simulation anal-
ysis is in good agreement with Veljkovics and Henzlers results.
The predicted values, however, are much higher than those of C.
Mrotzek and Maier. This may be caused by the fact that the satura-
tion concentration of oxygen (c*) is 0.109 mol/m3 in Maiers work
obtained through experimental data in microbial culture medium,
wherein oxygen solubility is mainly inuenced by the presence of
salts. In our work, c* (0.265 mol/m3 ) is based on Henrys law for the
assumption of pure water at 1 atm (20 C). It was consistent with
our CFD simulation results using the pure water c* to calculate kL a.
This CFD model could also be veried with the work of Chao Li
[23]. Therefore, the CFD simulation results are consistent with the
literature.
3.2. Hydrodynamic environment investigation
The morphology of the free surface (gasliquid interface)
formed in unbafed and bafed asks is shown in Fig. 1. The shear
stress distribution simulation results are shown in Fig. 3. As men-
tioned above, the shear stress consists of two different aspects: the
gradients of the uctuating velocity eld caused by turbulence (t ),
69
Correlation proposed

  g 3 1/3  d3 8/9  1 1/2  8/27 do d 1/2
2
kL a = 0.5 DF
 VL  d3 g g
 0.845
V
kL a = 0.032 n
VL
2/3 Vb N
kL a(A T ) with A = (V VL ) T= VL
60
A = 142, b = 0.463 and kL a = 0.0182 (A T )
kL a = 6.67 106 n1.16 V 0.83 d00.38 d1.92
OTRmax 1
kL a = = n0.84 V 0.84 d00.27 d1.25
c c
and the velocity gradient at two sides of the cells by turbulence (d ).
The results demonstrated that the highest stress is contributed by
d in bafed and un-bafed shaken asks, which is approximately
3 times higher compared with t . The total shear stress (both d
and t ) distributions for bafed and un-bafed asks operated at
200 rpm and 150 ml lling volume are shown in Fig. 3c. The shear
force distribution in un-bafed asks is much broader than that
in bafed asks. At the same operational condition, a bafed ask
can produce higher shear force than an un-bafed one. The shear
environment under different operating conditions (Fig. 4) shows
that the shear force increases as the rotation speed increases, and
it decreases with increasing lling volume.
Several shear sensitivity studies have reported that the unsta-
ble effects on plant cells of hydrodynamic stress include not only
the average shear force but also the hydrodynamic heterogene-
ity, dened by max = max /ave [5,47]. T These are quite important
when taking the local ow conditions and microscopic structure
of the ow into consideration when scaling-up a process [15,48].
However, most of the sensitivity studies on plant cells have just
focused on the average behavior of the ow, such as average
shear stress and total energy dissipation. These variables cannot
be applied well to other mixing devices or other scale bioreac-
tor systems because the hydrodynamic heterogeneity conditions
always change greatly between different mixing devices and scales.
Therefore, this heterogeneity (max ) is difcult to determine accu-
rately through experiments. Thus, values of max from 10 to 100
in a stirred tank have been reported, where a typical value of 30
is observed [47,49]. CFD analysis could overcome the limitation of
70 Y. Liu et al. / Biochemical Engineering Journal 113 (2016) 6676

Table 2
Summary of the experiments, including Re, ave, t,ave d,ave and max/ ave.

Run No Filling Rotation Re ave t ,ave d ,ave max /ave

volume speed

ml rpm w kg-1 Pa Pa
u1 100 200 4 105 0.37 0.43 1.31 13.31
u2 150 200 4 105 0.27 0.35 1.03 14.09
u3 200 200 4 105 0.21 0.31 0.86 15.87
u4 150 150 3 105 0.07 0.20 0.46 12.75
u5 150 115 3 105 0.02 0.11 0.21 12.20
b1 150 150 3 105 0.15 0.32 0.81 10.93
b2 200 200 4 105 0.39 0.47 1.41 16.71
b3 150 200 4 105 0.59 0.57 1.85 18.82

u indicates un-bafed shaken ask. b indicates bafed shaken ask.

Re = l nds 2 /l , ds is the ask diameter.

Table 3
Input values for s, O simulation.

Parameter Unit Value Reference

1
max h 0.0120 Measured
C*O2 mmol/kg 0.2650 Henrys law
Ko2 mmol/kg 0.0023 Measured
Ks g/L 2.6 W.M. van Gulik. [51]
qomax mmol/(h dwg) 0.44 Measured
qsmax g/(h dwg) 0.03 Measured
kL a h1 15.54122.02 CFD result
max h-1 0.0133 Measured

environment is more heterogeneous, and also max will increase

with increasing shake speed and as the lling volume decreases.

3.3. Elimination of the inuence of oxygen and sugar uptake rate

To quantify and evaluate the effect of shear forces on plant cell

growth, O CO /(kO + CO ) and s = Cs/(ks + Cs) were used to identify
the inuence of oxygen and substrate uptake rate on cell growth
and viability [50]. Co is the concentration of oxygen in liquid, and
it was calculated by the maximum oxygen transfer rate (OTR) and
maximum specic oxygen uptake rate as given in Equations 8, 9 and
10, with detailed parameters shown in Table 3. We consider that the
lowest oxygen uptake rate conditions (kL a = 15.54 h1 ) were from
0 to 120 h with a change of O 0.98920.9713 and s change from
0.9090 to 0.8505.
Under the middle oxygen uptake rate conditions
(kL a = 37.13 h1 ), O changed from 0.9904 to 0.9874 and s
changed from 0.9088 to 0.8502. Therefore, the inuence of oxygen
and sugar uptake rates will be almost constant during this culture
period, and the specic growth rate will be primarily inuenced
by hydrodynamic stress.

OTR = kL a(c c) (8)

cO
OUR = qo,max ( )X (9)
cO + kO
dC
= OTR OUR (10)
dt
Fig. 3. Comparison of the distributions of the hydrodynamic stress d and t in
(a) bafed ask (b) unbafed ask at 200 rpm and 150 ml lling volume, (c) the
3.4. Effects of shear stress on cell growth and metabolism
distributions of the total hydrodynamic stress in un-bafed and bafed shaken ask.

Time courses of dry weight, cell capacitance, residual sugar, con-

unknown local ow conditions. The results (Table 2) for shaken
asks showed that the value of max = max /ave is as large as 1018,
which means that the shear distribution is more uniform than that
in a stirred tank (in this paper, we dene max as the local and maxi-
mum energy dissipation rate in the top 1 percentage of the volume).
In the same operational conditions, max in a bafed ask will be
higher than in an un-bafed ask, which illustrates that the shear
ductivity, pH and mitochondrial activity are presented in Fig. 5. The
control culture is the average of four low shear condition cultures
in which cell growth and viability did not show differences. The
error bars are standard deviations from biological replicates of each
culture. The results showed that the cell growth rate was greatly
inhibited as the shear force increased (Fig. 5a). The highest biomass
concentration of 8.5 g/L was obtained at 120 h under the low shear
 Y. Liu et al. / Biochemical Engineering Journal 113 (2016) 6676
Fig. 4. Stress values (both d and t)calculated via CFD under various operate condition, lling volume (V) is 2050% of nominal volume, shaking frequency (n) is in the range
of 115220 rpm, and shaking diameter is 50 mm, (a) un-bafed ask (b) bafed ask.
condition (<0.41 Pa), which was 2.8 times higher than that under
the highest shear level (3.0 g/L) when the shear force reached
2.42 Pa (150 ml, 200 rpm). These can also be demonstrated from the
utilization of sugar and major salts, as the residual sugar concen-
tration and broth conductivity decreased with the cell growth rate
(Fig. 5c,d). Fig. 5b shows a time course of cell capacitance, which is
a more accurate and conventional biomass determination method.
Markx et al. [52] found that capacitance can be used in cell shear
sensitivity studies because the determined capacitance is depen-
dent on the potential and integrity of the membrane. Therefore,
capacitance is used to evaluate the percent of membrane-damaged
cells under different shear stress conditions at the initial culture
phase. Statistical analysis revealed that more than 16.5% of cells
lost membrane integrity when the average shear stress increased to
2.42 Pa. The pH hardly differed under various shear force conditions
(Fig. 5e). Viability determined according to mitochondrial activ-
71
ity, normalized to the results of the low shear condition (Fig. 5f),
showed that viability decreased more than 75% during the former
4 days, and the viability loss was mostly dependent on the shear
force level. However, viability surprisingly recovered to the nor-
mal level after a 90-h culture process. These ndings suggest that
under our shear conditions, 90 h is the turning point for cell growth,
at which the specic growth rate and substrate consumption rate
recover to the normal level. These phenomena could be realized as
the adaptation of plant cells to the unfriendly high shear condi-
tions [19,53]. In animal cell shear studies, a similar phenomenon
was also found [54].
3.5. Effect of shear stress on cell aggregation
Cell aggregation is usually considered to be one of the most
important characteristics in plant cell cultivation [7,55]. In this
72 Y. Liu et al. / Biochemical Engineering Journal 113 (2016) 6676

Fig. 5. Comparison of (a) dry weight (b) permittivity (c) total sugar (d) conductivity (e) cell viability (TTC) (f) pH measured during cultivation for average shear force ave .
Control () 1.17 Pa (--), 1.39 Pa ( ), 1.74 Pa ( ), 1.88 Pa ( ) and 2.42 Pa ( ).

study, the effects of shear stress on C. tinctorius L. cell aggrega-
tion were analyzed by FBRM (Fig. 6). As observed from Fig. 6a,
the average aggregation diameter increases with time until reach-
ing the highest diameter of 280 m. Meanwhile, the aggregation
diameter showed great differences under various shear environ-
ments during the initial culture phase of 75 h. During this period,
the cell aggregation diameter increased from 125 m to 300 m
under the lowest shear stress of 1.17 Pa, whereas when shear stress
increased to 2.42 Pa, the highest particle diameter decreased to
230 m, which is 25% lower than that at 1.17 Pa. However, the
diameter of the particles tends to remain uniform after 8090 h of
shear treatment. The distribution of cell aggregation size at 100 h
(Fig. 6b) indicates that there was hardly any difference under var-
ious hydrodynamic environments. These results suggest that the
aggregations of C. tinctorius L. can also become adapted to a
high shear environment in morphological characterization. These
results differ from previously reported work [10,12,25], in which
cell aggregates have a decreasing tendency with increasing shear
stress. Such as Morinda citrifolia cells were studied by shearing the
suspended cells in a well-dened capillary tube, and the average
 Y. Liu et al. / Biochemical Engineering Journal 113 (2016) 6676
Fig. 6. (a) average cell aggregate size over culture time and (b) the distribution of cell aggregations size at 100 h under different hydrodynamic stress values (100 h), 1.17 Pa
(--), 1.39 Pa ( ), 1.74 Pa ( ), 1.88 Pa ( ) and 2.42 Pa ( ).
shear force was approximately 40 Pa [10]. the results showed that
the aggregation length distribution was shifted toward lower val-
ues. In another study on C. tinctorius L. cultivation [25], a shear
environment was produced by different agitation speeds elevated
from 200 to 500 rpm for 24 h in a stirred reactor. The cells average
aggregation diameter changed from 276 m to 139 m, a greater
decrease in diameter than was observed in our experiment.
3.6. Stress threshold determination
To quantitatively determine the stress threshold, the maximum
specic growth rate (max ) was represented as a function of the
average and maximum shear force (Fig. 7). In addition, Students
t-test was used to identify differences between cultivations per-
formed at different stress levels. All data were compared with the
control culture, where p-values below 0.1 were considered dif-
ferent (labeled with one star) and those lower than 0.05 were
considered signicantly different (labeled with two stars). The sta-
73
tistical results showed that the rst ve lower stresses showed
no signicant difference, whereas the last two points at higher
stress levels resulted in p-values lower than 0.05, which is a sig-
nicant difference. Based on the experiments performed at various
stress levels, a polynomial tting simulation (function) was cre-
ated to evaluate the threshold of the shears effect. The stress
threshold was dened with a decreased specic growth rate no
less than 95% of max . The results demonstrate that C. tinctorius
L cell growth would not show detectable differences under low
hydrodynamic stress with average and maximum shear levels of
0.55 Pa (0.06 w/kg) and 4.00 Pa (0.93 w/kg), respectively. However,
the growth will be strongly impacted in higher shear environments
(>1.57 Pa, 50% max ).
Most previous research has made empirical correlations based
on parameters of rotation speed or impeller tip speed to assess
the effects of hydrodynamic stress. Obviously, these parameters
are always scale-dependent and difcult to evaluate quantitatively.
Moreover, the critical shear stress also varies greatly by cell line,
74 Y. Liu et al. / Biochemical Engineering Journal 113 (2016) 6676
Fig. 7. Specic growth rate (max ) as a function of (a) the average shear force various and (b) maximum shear force.
age, ow type and frequency of exposure time [5]. In early studies
of hydrodynamic stress on C. tinctorius L cells, Takedas research
indicated that some shear induced ATP and NAD(P)H reduction and
a cytosolic calcium content increase at the level of 0.52.3 w/kg
energy dissipation rate [24,25]. Dunlop and co-workers [12] per-
formed comprehensive studies to investigate the effects of both
laminar and turbulent conditions using rotational viscometers on
plant cell suspension culture. A wide range of stress indicators was
employed to clearly identify a hierarchy of cellular responses. How-
ever, as with most previous studies, it is difcult to compare the
results due to the uid analytical technique, which is presented as
a function of cumulative energy dissipation (J/m3 ). Hence, some
comparison has been added with a recent study on animal cells.
In a CHO cell scaled-down hydrodynamic stress study, the thresh-
old value of the average energy dissipation rate was determined as
0.4 w/kg in batch culture, beyond which productivity will decline.
Moreover, a transcriptomic stress response was found [16], which is
higher than our result (0.06 w/kg). This illustrates that C. tinctorius L
cells are as sensitive to shear stress as animal cells or more so, even
though they have a cell wall. Furthermore, in another long-term
shear study, threshold maximum shear force values of 32.4 4.4 Pa
and 25.2 2.4 Pa for CHO and Sp2/0 were reported by comparing
cell growth and productivity rates in an external loop shear device
[54], where a signicant difference was found. However, we believe
that the major source of this difference is related to the frequency
and period of cell exposure to hydrodynamic stress. The exposure
period was set to 90 s to simulate the circulation time in a 5000-L
bioreactor in Neunstoecklins work, but the circulation time in a
shaken ask is approximately 0.51.0 s [56]. As the exposure time
is relatively short and followed by a very long period for cells to
recover, it can be understood that our result is much lower under
both higher exposure time and frequency.
4. Conclusion
In the present work, we developed a shear force study system
based on CFD technology in commonly used shaken ask biore-
actors under various operating conditions. A different approach
 Y. Liu et al. / Biochemical Engineering Journal 113 (2016) 6676
was used to evaluate stress damage to plant cell lines. Compared
with others methods that only use energy dissipation rates to eval-
uate shear force, the approaches applied in the present research
could better characterize the effect of large size plant aggregations.
Moreover, sub-lethal effects caused by various hydrodynamics
on C. tinctorius L. cells were effectively evaluated. The investiga-
tions results indicated that specic cell growth rates were greatly
inhibited and that the lag phase was seriously postponed with
an increase in shear force levels. A similar effect on cell sub-
strate consumption rates (sugar and salt) could also be observed.
Although viability decreased rapidly under the shear treatment, it
could recover to normal levels after a period of time, which means
that C. tinctorius L. cells could become adapted to certain shear
conditions. Moreover, the cell aggregations also tended to be uni-
form under various hydrodynamic stress levels after the viability
recovery. Finally, a threshold was observed at an average and max-
imum shear stress of approximately 0.55 Pa (0.06 w/kg) and 4.00 Pa
(0.93 w/kg) according to the inuence on cell growth. The method
used in this work can be applied to quantitatively study other plant
cells. Quantitative evaluation of the effects of shear force can facil-
itate industrial process design and lead to more rational scale-up
of C. tinctorius L. suspension cultures.
Acknowledgements
This work was nancially supported by the National High Tech-
nology Research and Development Program (2015AA021005), and
973 Program No. 2013CB733600. the Royal DSM and partially sup-
ported by NOW-MoST Joint Program (2013DFG32630). We also
thank pharmacy corporation Practical Bio Co., Ltd. (DALIAN, China)
for donating the industrial strain.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bej.2016.06.001.
References
[1] K.M. Davies, S.C. Deroles, Prospects for the use of plant cell cultures in food
biotechnology, Curr. Opin. Biotechnol. 26 (2014) 133140.
[2] S.-Y. Han, H.-X. Li, X. Ma, K. Zhang, Z.-Z. Ma, P.-F. Tu, Protective effects of
puried safower extract on myocardial ischemia in vivo and in vitro,
Phytomedicine 16 (2009) 694702.
[3] N. Hanagata, I. Karube, Red pigment production by Carthamus tinctorius cells
in a two-stage culture system, J. Biotechnol. 37 (1994) 5965.
[4] T.M. Chong, M.A. Abdullah, N.M. Fadzillah, O.M. Lai, N.H. Lajis, Jasmonic acid
elicitation of anthraquinones with some associated enzymic and
non-enzymic antioxidant responses in Morinda elliptica, Enzyme Microb.
Technol. 36 (2005) 469477.
[5] P.M. Kieran, D.M. Malone, P.F. MacLoughlin, Effects of hydrodynamic and
interfacial forces on plant cell suspension systems, Adv. Biochem. Eng.
Biotechnol. 67 (2000) 139177.
[6] T.K. Huang, K.A. McDonald, Bioreactor systems for in vitro production of
foreign proteins using plant cell cultures, Biotechnol. Adv. 30 (2012) 398409.
[7] T.K. Huang, K.A. McDonald, Bioreactor engineering for recombinant protein
production in plant cell suspension cultures, Biochem. Eng. J. 45 (2009)
168184.
[8] C.B.E.J.B. Joshi, M.S. Patole, Role of hydrodynamic shear in the cultivation of
animal, plant and microbial cells, Biochem. Eng. J. 62 (1995) 121141.
[9] P.M. Doran, Design of mixing systems for plant cell suspensions in stirred
reactors, Biotechnol. Progr. 15 (1999) 319335.
[10] P.M. Kieran, H.J. ODonnell, D.M. Malone, P.F. MacLoughlin, Fluid shear effects
on suspension cultures of Morinda citrifolia, Biotechnol. Bioeng. 45 (1995)
415425.
[11] P.F. MacLoughlin, D.M. Malone, J.T. Murtagh, P.M. Kieran, The effects of
turbulent jet ows on plant cell suspension cultures, Biotechnol. Bioeng. 58
(1998) 595604.
[12] E.H. Dunlop, P.K. Namdev, M.Z. Rosenberg, Effect of uid shear forces on plant
cell suspensions, Chem. Eng. Sci. 49 (1994) 22632276.
[13] D.D. Sowana, D.R.G. Williams, E.H. Dunlop, B.B. Dally, B.K. ONeill, D.F.
Fletcher, Turbulent shear stress effects on plant cell suspension cultures,
Chem. Eng. Res. Des. 79 (2001) 867875.
75
[14] F. Garca Camacho, J.J. Gallardo Rodrguez, A. Snchez Mirn, M.C. Cern
Garca, E.H. Belarbi, E. Molina Grima, Determination of shear stress thresholds
in toxic dinoagellates cultured in shaken asks: implications in bioprocess
engineering, Process Biochem. 42 (2007) 15061515.
[15] A.W. Nienow, Re development of a scale-down model of hydrodynamic stress
to study the performance of an industrial CHO cell line under simulated
production scale bioreactor conditions [Sieck, J.B. Cordes, T., Budach, W.E.,
Rhiel, M.H., Suemeghy, Z., Leist, C., Villiger, T.K., Morbidelli, M., Soos, M., 2013.
journal of biotechnology 164, 4149], J. Biotechnol. 171 (2014) 8284.
[16] J.B. Sieck, T. Cordes, W.E. Budach, M.H. Rhiel, Z. Suemeghy, C. Leist, T.K.
Villiger, M. Morbidelli, M. Soos, Development of a Scale-Down Model of
hydrodynamic stress to study the performance of an industrial CHO cell line
under simulated production scale bioreactor conditions, J. Biotechnol. 164
(2013) 4149.
[17] M. Keler, H.G. ten Hoopen, J. Heijnen, S. Furusaki, O uptake rate
measurements as a novel tool to study shear effects on suspended strawberry
cells, Biotechnol. Tech. 11 (1997) 507510.
[18] C.-H. Ho, K.A. Henderson, G.L. Rorrer, Cell damage and oxygen mass transfer
during cultivation of Nicotiana tabacum in a stirred-tank bioreactor,
Biotechnol. Progr. 11 (1995) 140145.
[19] J.J. Meijer, H.J.G. ten Hoopen, Y.M. van Gameren, K.C.A.M. Luyben, K.R.
Libbenga, Effects of hydrodynamic stress on the growth of plant cells in batch
and continuous culture, Enzyme Microb. Technol. 16 (1994) 467477.
[20] J.E. Schlatmann, A.M. Nuutila, W.M. Van Gulik, H.J.G. ten Hoopen, R.
Verpoorte, J.J. Heijnen, Scaleup of ajmalicine production by plant cell cultures
of Catharanthus roseus, Biotechnol. Bioeng. 41 (1993) 253262.
[21] J. Buchs, U. Maier, C. Milbradt, B. Zoels, Power consumption in shaking asks
on rotary shaking machines: i. Power consumption measurement in unbafed
asks at low liquid viscosity, Biotechnol. Bioeng. 68 (2000) 589593.
[22] H. Zhang, W. Williams-Dalson, E. Keshavarz-Moore, P.A. Shamlou,
Computational-uid-dynamics (CFD) analysis of mixing and gasliquid mass
transfer in shake asks, Biotechnol. Appl. Biochem. 41 (2005) 18.
[23] C. Li, J.-Y. Xia, J. Chu, Y.-H. Wang, Y.-P. Zhuang, S.-L. Zhang, CFD analysis of the
turbulent ow in bafed shake asks, Biochem. Eng. J. 70 (2013) 140150.
[24] T. Takeda, T. Kitagawa, Y. Takeuchi, M. Seki, S. Furusaki, Metabolic responses
of plant cell culture to hydrodynamic stress, Can. J. Chem. Eng. 76 (1998)
267275.
[25] T. Takeda, M. Seki, S. Furusaki, Hydrodynamic damage of cultured cells of
Carthamus tinctorius in a stirred tank reactor, J. Chem. Eng. Jpn. 27 (1994)
466471.
[26] N. Gregoriades, J. Clay, N. Ma, K. Koelling, J.J. Chalmers, Cell damage of
microcarrier cultures as a function of local energy dissipation created by a
rapid extensional ow, Biotechnol. Bioeng. 69 (2000) 171182.
[27] T. Tanzeglock, M. Soos, G. Stephanopoulos, M. Morbidelli, Induction of
mammalian cell death by simple shear and extensional ows, Biotechnol.
Bioeng. 104 (2009) 360370.
[28] I. Abu-Reesh, F. Kargi, Biological responses of hybridoma cells to
hydrodynamic shear in an agitated bioreactor, Enzyme Microb. Technol. 13
(1991) 913919.
[29] J.F. Petersen, L.V. McIntire, E.T. Papoutsakis, Shear sensitivity of cultured
hybridoma cells (CRL-8018) depends on mode of growth, culture age and
metabolite concentration, J. Biotechnol. 7 (1988) 229246.
[30] C. Smith, P. Greeneld, D. Randerson, A technique for determining the shear
sensitivity of mammalian cells in suspension culture, Biotechnol. Tech. 1
(1987) 3944.
[31] M. Mollet, R. Godoy-Silva, C. Berdugo, J.J. Chalmers, Acute hydrodynamic
forces and apoptosis: a complex question, Biotechnol. Bioeng. 98 (2007)
772788.
[32] N. Ma, K.W. Koelling, J.J. Chalmers, Fabrication and use of a transient
contractional ow device to quantify the sensitivity of mammalian and insect
cells to hydrodynamic forces, Biotechnol. Bioeng. 80 (2002) 428437.
[33] S. Werner, J. Olownia, D. Egger, D. Eibl, An approach for scale-up of
geometrically dissimilar orbitally shaken single-use bioreactors, Chem. Ing.
Tech. 85 (2013) 118126.
[34] D.-Y. Peng, D.B. Robinson, A new two-constant equation of state, Ind. Eng.
Chem. Fundam. 15 (1976) 5964.
[35] J.C. Lamont, D.S. Scott, An eddy cell model of mass transfer into the surface of
a turbulent liquid, AIChE J. 16 (1970) 513519.
[36] R. Sorg, T. Tanzeglock, M. Soos, M. Morbidelli, A. Perilleux, T. Solacroup, H.
Broly, Minimizing hydrodynamic stress in mammalian cell culture through
the lobed Taylor-Couette bioreactor, Biotechnol. J. 6 (2011) 15041515.
[37] J.O. Hinze, Fundamentals of the hydrodynamic mechanism of splitting in
dispersion processes, AIChE J. 1 (1955) 289295.
[38] S.B. Pope, Turbulent Flows, Cambridge University Press, 2000.
[39] T. Holland, D. Blessing, S. Hellwig, M. Sack, The in-line measurement of plant
cell biomass using radio frequency impedance spectroscopy as a component
of process analytical technology, Biotechnol. J. 8 (2013) 12311240.
[40] G.H. Markx, C.L. Davey, D.B. Kell, P. Morris, The dielectric permittivity at radio
frequencies and the bruggeman probe: novel techniques for the on-line
determination of biomass concentrations in plant cell cultures, J. Biotechnol.
20 (1991) 279290.
[41] L.E. Towill, P. Mazur, Studies on the reduction of 2,3,5-triphenyltetrazolium
chloride as a viability assay for plant tissue cultures, Can. J. Bot. 53 (1975)
10971102.
[42] H.J. Henzler, M. Schedel, Suitability of the shaking ask for oxygen supply to
microbiological cultures, Bioprocess. Eng. 7 (1991) 123131.
76 Y. Liu et al. / Biochemical Engineering Journal 113 (2016) 6676
[43] U. Maier, J. Bchs, Characterisation of the gasliquid mass transfer in shaking
bioreactors, Biochem. Eng. J. 7 (2001) 99106.
[44] H. Nikakhtari, G.A. Hill, Modelling oxygen transfer and aerobic growth in
shake asks and well-mixed bioreactors, Can. J. Chem. Eng. 83 (2005)
493499.
[45] M.U., GasFlssigkeitsStofftransfer im Schttelkolben, RWTH Aachen
University, 2002.
[46] C. Mrotzek, T. Anderlei, H.-J. Henzler, J. Bchs, Mass transfer resistance of
sterile plugs in shaking bioreactors, Biochem. Eng. J. 7 (2001) 107112.
[47] A. Nienow, Reactor engineering in large scale animal cell culture,
Cytotechnology 50 (2006) 933.
[48] A.W. Nienow, W.H. Scott, C.J. Hewitt, C.R. Thomas, G. Lewis, A. Amanullah, R.
Kiss, S.J. Meier, Scale-down studies for assessing the impact of different stress
parameters on growth and product quality during animal cell culture, Chem.
Eng. Res. Des. 91 (2013) 22652274.
[49] C.J. Hewitt, A.W. Nienow, The scale-up of microbial batch and fed-batch
fermentation processes, Adv. Appl. Microbiol. (2007) 105135 (Academic
Press).
[50] J. Morchain, J.-C. Gabelle, A. Cockx, A coupled population balance model and
CFD approach for the simulation of mixing issues in lab-scale and industrial
bioreactors, AIChE J. 60 (2014) 2740.
[51] W.M. van Gulik, H.J.G. ten Hoopen, J.J. Heijnen, Kinetics and stoichiometry of
growth of plant cell cultures of Catharanthus roseus and Nicotiana tabacum in
batch and continuous fermentors, Biotechnol. Bioeng. 40 (1992) 863874.
[52] G.H. Markx, H.J.G. ten Hoopen, J.J. Meijer, K.L. Vinke, Dielectric spectroscopy
as a novel and convenient tool for the study of the shear sensitivity of plant
cells in suspension culture, J. Biotechnol. 19 (1991) 145157.
[53] A.H. Scragg, E.J. Allan, F. Leckie, Effect of shear on the viability of plant cell
suspensions, Enzyme Microb. Technol. 10 (1988) 361367.
[54] B. Neunstoecklin, M. Stettler, T. Solacroup, H. Broly, M. Morbidelli, M. Soos,
Determination of the maximum operating range of hydrodynamic stress in
mammalian cell culture, J. Biotechnol. 194 (2015) 100109.
[55] M.E. Kolewe, M.A. Henson, S.C. Roberts, Analysis of aggregate size as a process
variable affecting paclitaxel accumulation in Taxus suspension cultures,
Biotechnol. Prog. 27 (2011) 13651372.
[56] R.-K. Tan, W. Eberhard, J. Bchs, Measurement and characterization of mixing
time in shake asks, Chem. Eng. Sci. 66 (2011) 440447.