Appl Microbiol Biotechnol (1997) 48: 149154

Springer-Verlag 1997

ORIGINAL PAPER

C. Bohme M.-B. Schroder H. Jung-Heiliger

J. Lehmann

Plant cell suspension culture in a bench-scale fermenter

with a newly designed membrane stirrer for bubble-free aeration

Received: 22 October 1996 / Revised version: 11 March 1997 / Accepted: 14 March 1997

Abstract In this paper, tests of an optimized membranestirrer geometry for bubble-free aeration of a plant cell
suspension culture are described. Cell attachment and
clogging of a previously described system [Piehl et al.
(1988) Appl Microbiol Biotechnol 29:456461] led to the
development of a new stirrer. The volumetric oxygen
transfer capacity has been measured in aqueous medium. The mass transfer coecient, kla, was 3.75 h)1 at
25 C and at a stirrer speed of 34 rpm. The overall oxygen transfer capacity was investigated with a suspension culture of Aesculus hippocastanum. It was shown
that the oxygen mass transfer was sucient even at the
maximum biomass of 1012 g dry weight/l, which was
obtained by using this system. Furthermore, special attention was given to medium components like C and N
sources, to avoid growth limitation due to a shortage of
nutrients.

Introduction
When plant cell suspensions are cultivated in bubbleaerated fermenters, the otation of cells to the surface
results in an inhomogeneous biomass distribution and
C. Bohme (&) J. Lehmann
AG Zellkulturtechnik, Technische Fakultat,
Universitat Bielefeld, Postfach 100131,
D-33501 Bielefeld, Germany
Tel. (49) 521 106 6322; Fax (49) 521 106 6328
e-mail: cbo@zellkult.techfak.uni-bielefeld.de
M.-B. Schroder1
Institut fur Lebensmittelchemie, TU Braunschweig,
Schleinitzstr. 20, D-38106 Braunschweig, Germany
H. Jung-Heiliger
Celler Panzen- und Gewebelabor GmbH,
Postfach 1246, D-29202 Celle, Germany
Present address:
1
Forschungsanstalt Geisenheim, Institut fur Biologie,
Fachgebiet Botanik; Von-Lade-Str. 1,
D-65366 Geisenheim, Germany

the formation of foam. The accumulated biomass in the

foam experiences undened and uncontrolled conditions,
which may aect the whole fermentation process. For
fermentation in homogeneous systems, a bubble-free
aeration is preferable to avoid these disadvantages. For
this purpose a bubble-free aerated fermenter system with
an internal membrane stirrer was constructed for mammalian cell culture (Lehmann et al. 1987), which was
successfully applied for bench- and pilot-scale fermenters.
The membrane-stirrer reactor was also used for a suspension culture of Thalictrum rugosum (Piehl et al. 1988).
The aeration system consisted of a stirrer equipped
with a hydrophobic, microporous hollow-bre membrane, driven from the bottom by an external magnetic
stirrer. However, when used with a plant cell culture, the
membrane coil can get clogged by cells attached to the
membrane surface, which aects the oxygen-transfer
performance.
In plant cell suspension cultures aggregates of cells
are often formed, which adhere to the membrane,
clogging and covering the membrane coil. Therefore,
there was a need to construct a membrane stirrer that
would not allow plant cells to adhere to the membrane
or would wash adhering cell clusters from its surface.

Materials and methods

Membrane stirrer
For bubble-free agitation and aeration of a plant cell suspension,
the same principle was used that had already been proved for animal cell cultures (Lehmann et al. 1987).
A hollow-bre membrane (diameter 2.6 mm) was installed on a
stirrer that was exibly xed to the top plate of the reactor vessel.
The polypropylene membrane was hydrophobic and microporous
with a pore diameter of 0.2 lm. A magnet was integrated in the
lower end of the stirrer, which allowed movement by an external
magnetic stirrer under the reactor vessel. The agitation of the culture broth was carried out by the tumbling movement of the
membrane stirrer, with a low stirrer speed of approximately 3040
rpm to ensure good mixing at low shear stress.
To prevent clogging of the stirrer arrangement by cell clusters,
the membrane coil was mounted on a ring support where the

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windings of the membrane were arranged in a widely spaced array.
The gap between the windings was greater than the diameter of the
biggest cell clusters.
The chosen distance was 2.5 mm, allowing a laminar ow of the
culture broth around the membrane bre surface (Fig. 1).

Exhaust

Two-litre reactor system

Inoculum

A 2-l fermenter (Biostat BF, B. Braun Melsungen, Germany),

equipped for bubble-free aeration, was used as shown in Fig. 2.
The dissolved oxygen concentration was set at 30% air saturation
and was measured by a PO2 sensor connected with a digital control
unit, which controlled the gas-mixing station. The pH was measured and displayed but not controlled by the digital control unit.
The cultivation was performed as batch culture with replacement of
used medium when nutrients were depleted. For this purpose, the
supernatant above the sediment was removed and replaced with
fresh medium after sedimentation of the biomass. The glass culture
vessel was covered with a foil of aluminium to cultivate the cells in
the dark. The temperature was set at 25 C, the stirrer speed at
34 rpm.

To determine the volumetric mass-transfer coecient, kLa of the

reactor system, the dynamic method in a water/air system was
applied (Wang et al. 1979).

N2

CO2

pO2

pH

Digital control unit

Temperature control

Magnetic stirrer

Medium

The oxygen transfer rate is proportional to the driving force

c* ) cL of the mass transfer according to:
dc
kL a c cL
1
dt
where OTR (mg O2 ml)1 h)1) = volumetric rate of oxygen transfer, kL (cm/h) = liquid phase mass-transfer coecient, a = A/V
(cm2/ml) = specic interfacial area, c* (mg O2/ml) = equilibrium
oxygen concentration in the liquid phase, cL (mg O2/ml) = dissolved oxygen concentration in the liquid phase, and t (h) = time.
To evaluate the oxygen transfer performance of the membrane
stirrer in the given reactor system, the kLa value is the characterising quantity. For the determination of kLa the integrated form of
Eq. 1 was employed:
  
c
ln 
kL at
2
c cL

5 mm

OTR

Gas out

195 mm

When the left-hand term of Eq. 2 was plotted against time, the kLa
resulted from the slope. The slope was calculated using linear regression. In order to determine cL, oxygen was rst stripped o
with nitrogen, and aeration with air was switched on with a dened
mass ow (0.18 mol/h). The increasing PO2 was measured with an
amperometric polarographic oxygen electrode (Mettler Toledo,
Germany). The calibration was done with the air saturation concentration. All measurements were carried out in water (2 l) at
25 C, with the same headspace pressure in the fermenter and a
stirrer speed of 34 rpm. The air saturation concentration, c*, at
25 C was 8.122 mg/l, the membrane surface A was 0.049 m2.

N
8 mm

Air

Gas mixing station

Fig. 2 Experimental set-up of the 2-l bioreactor system equipped with

a membrane stirrer for bubble-free aeration

Measurement of oxygen-transfer capacity

Gas in

O2

Sample port

55 mm

Fig. 1 Membrane stirrer developed for plant cell suspension culture

(membrane is partly shown)

Plant material
For testing the newly designed reactor system with a plant suspension culture, Aesculus hippocastanum cells were chosen, because
this species produces substances of pharmaceutical importance e.g.
the triterpenoid aescin.
In 1992 the explants were excised from horsechestnut seeds
(cotyledones) and cultivated on agar plates with modied Murashige-Skoog (Murashige and Skoog, 1962) medium. For callus
induction, the phytohormones benzylaminopurine (0.45 mg/l) and
naphthaleneacetic acid (3 mg/l) were added to the medium. To
obtain a cell suspension, friable calli were isolated and transferred
into liquid medium with an altered hormone composition. The
cultures were maintained in the dark at 22 C and 27 C respec-

151
tively. The cell suspension obtained was cultivated in 250-ml conical asks containing 100 ml suspension on a rotary shaker at
100 rpm at 25 C. The subcultivation interval lasted 10 days.
SuperSpinner culture
The SuperSpinner is a 1-l spinner ask recently developed in this
institute: it has internal membrane aeration and is used for mammalian cell culture (Heidemann et al. 1994). The membrane coil
was moved by a magnetic stirrer at the bottom. For aeration the
SuperSpinner system consisted of a membrane pump for air supply
and was placed in an air-conditioned room. This system also
worked with plant cell cultures. The 2-l fermenter was inoculated
from conical asks or from SuperSpinner cultures.
Medium composition
For fermentation and shake-ask culture, a Murashige-Skoogbased medium containing the phytohormones indoleacetic acid
(0.8 mg/l), naphthaleneacetic acid (3.0 mg/l) and benzylaminopurine (0.45 mg/l) as well as three amino acids (Gly, Gln, Ser) was
used.
Estimation of culture parameters
Growth The biomass was determined as fresh weight and dry weight
(freeze drying). The sedimentation volume was measured after a
dened time of settling (15 min). For testing cell viability the vital
dye uorescein diacetate was used.
Amino acids (Buntemeyer et al. 1991) Amino acids were quantied
by reverse-phase HPLC (Beckmann, Ultrasphere ODS C18). For
detection with a uorescence detector the samples were derivatised
with O-phthaldialdehyde before being applied to the column. For
elution, a binary buer system (methanol/acetate buer) with increasing polarity was used.

A was routinely applied for mammalian cell cultures and

also for T. rugosum plant cell cultures (Piehl et al. 1988);
second: stirrer B, as described above, was developed for
coarse plant cell suspensions.
Stirrer A showed strong clogging after fermentation.
The plant material clogged the space between the hollow
bres almost completely (Fig. 3a). Only a few areas
where there was considerable distance between the hollow bres were not aected. This nding nally led to
the development of a stirrer that allowed the membrane
to be mounted with greater distances between the
windings.
On stirrer B (Fig. 3b) no comparable membrane
clogging was observed, because of the widely spaced
membrane array. The membrane geometry prevented
cells or cell clusters from becoming attached to the
membrane. The measurement of the oxygen transfer
coecient kLa indicated a 42% higher value for stirrer
B, although the same length of membrane (6 m) was
mounted on both stirrers (Table 1). The narrow windings of stirrer A prevented ow over the whole perimeter, whereas the membrane of stirrer B allowed laminar
ow around the surface of the membrane bres over
nearly the whole length.
Provided that the nutrient supply is sucient, the
oxygen-transfer capacity of the aeration system is the
limiting factor in fermentation. Therefore, the maximum
attainable biomass is dependent on its oxygen demand,
expressed as the specic O2 consumption rate, qO2, of
the culture. The qO2 can be indirectly determined by
measuring the specic consumption rate of the C source,
when total oxidation to CO2 is assumed. In fermentations using glucose as the only C source, the specic

Anions An HPLC assay was also employed for anion NO3;

2
PO3
4 ; SO4 detection. A Wescan Anion/R (Polystyrol, N(CH3)3 )
column, and 5 mM p-hydroxybenzoic acid, adjusted to pH 8.5 with
LiOH, was used for elution.

Ammonium The samples were made alkaline with 10 M NaOH to

convert NH
4 into NH3. NH3 was detected by an ammonia detector
(WTW, NH 500/2 and WTW, pMX 2000/Ion).

Sugars An automatic glucose analyser (Yellow Springs Instruments, USA) was used for glucose determination. Sucrose was
hydrolysed at pH 4.4 and 55 C for 45 min by invertase (EC
3.2.1.26, from bakers yeast; Sigma) diluted in acetate buer. The
resulting glucose concentration was measured and, from these data,
the original sucrose concentration was calculated. Fructose was
determined with a D-glucose, D-fructose test kit (Boehringer AG
Mannheim, Germany).

Results
Membrane stirrer
A. hippocastanum fermentations were carried out with
two dierent membrane stirrer geometries. First: stirrer

Fig. 3 a Membrane stirrer A after Aesculus hippocastanum fermentation. b Membrane stirrer B after A. hippocastanum fermentation

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Table 1 Volumetric oxygen mass transfer coecient, kLa, of the
investigated membrane stirrers. The maximum O2 transfer rate was
calculated from kLa according to Eq. 1 (with cL = 0), for aeration
with air and oxygen
Stirrer geometry

Membrane stirrer A
Membrane stirrer B

kLa
(l/h)

2.64
3.75

Maximum O2 transfer
rate (mg l)1 h)1)
In air

In O2

21.4
30.5

102.6
145.7

glucose consumption rate, qGlc, was not constant. During the growth phase, qGlc ranged from 7.31 to
21.93 mg g dry weight)1 h)1 with an average value of
14.62 mg g dry weight)1 h)1. The stoichiometric conversion of one molecule of glucose to CO2 requires six
molecules of O2 and allows the calculation of qO2 from
the specic glucose-consumption rate. With this method
the mean qO2 value of a growing A. hippocastanum
suspension culture was calculated to be 15.61 mg g dry
weight)1 h)1. From these data the theoretical maximum
attainable biomass in the investigated 2-1 reactor system
could be estimated (Table 2). With air aeration it was
3.95 g dry weight/l and with pure oxygen (with the 4.78fold O2 transfer rate) 18.88 g dry weight/l, when minimal
O2 demand for growth is assumed. This means that up
to 18.88 g dry weight/l could be supplied with oxygen.
Biomass production
In the 2-l reactor system a biomass of 1012 g dry
weight/l was attained. (Figs. 4, 5a, 6). At this cell density
the sedimentation volume almost met the working volume of the fermenter, and the suspension became very
viscous. Above 11 g dry weight/l, the aeration control
was not able to hold the set-point concentration of 30%,
even when aeration was done with pure oxygen (Fig. 6).
This indicates that the oxygen uptake rate of the culture
passes the maximum oxygen transfer rate at the dissolved oxygen level of 30% air saturation. A decreasing
dissolved oxygen concentration, cL, increases the O2

Table 2 Calculation of the maximum attainable dry weight as a

function of the maximum oxygen transfer rate of the 2-l bioreactor
equipped with stirrer B (kLa = 3.75). The maximum dry weight
results from the quotient O2 transfer rate/qO2. The qO2 was calculated from the specic glucose consumption rates, qGlc, and
ranged between 7.31 and 21.93 mg g dry weight)1 h)1 during the
growth phase of the A. hippocastanum culture. The maximum O2
transfer rate in air was 30.5 and in O2 it was 145.7 mg O2 l)1 h)1
qGlc
(mg g dry wt)1 h)1)

qO2 (mg g
dry wt)1 h)1)

Dry weight (g/l)

Air

O2

7.31
14.62
21.93

7.72
15.61
23.33

3.95
1.95
1.31

18.88
9.3
6.5

Fig. 4 Sedimented biomass (after a settling time of 15 min) after 12

days of batch culture

transfer rate according to Eq. 1, and the maximum oxygen transfer capacity will be reached when cL is zero.
At very low dissolved O2, growth may be aected, because of kinetic limitation of O2 uptake.
However, the limiting factor of biomass production
with the A. hippocastanum culture examined was the
huge biomass itself, which caused high viscosity of the
cell suspension. The highly viscous cell suspension was
hard to agitate and led to a loss in cell viability.
Nutrient consumption
The nutrient consumption was monitored to optimize
the medium composition. Sucrose, the main C source in
the medium, decreased after inoculation, while the formation of glucose and fructose could be observed. When
sucrose was depleted, the other sugars also decreased
(Fig. 5b). Therefore, the overall sugar consumption had
to be calculated as the sum of sucrose, glucose and
fructose. According to this nding, sucrose was substituted by glucose in all following fermentations without
negative eects on growth.
The appearance of glucose and fructose may be due
to the hydrolysis of sucrose by an extracellular invertase
and is a well-known phenomenon in plant cell culture
(Schmitz and Lorz 1988).
In order to ensure that the culture is not limited by
macronutrients (particularly N, P, and S sources), the
3
2
NO
concentrations were determined in
3 ; PO4 ; SO4
preliminary experiments (Fig. 7). Phosphate was depleted after 5 days, nitrate after 10 days and ammonia
mainly after 13 days of batch culture. Also the C sources

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Fig. 6 A. hippocastanum fermentation with the new membrane

stirrer B

Fig. 7 A. hippocastanum batch culture. Nutrient consumption of N, P

and S sources

Fig. 5ac A. hippocastanum fermentation with the new membrane

stirrer B. a Biomass, b sugars, c amino acids

(sucrose, fructose, glucose) were depleted after 13 days.

Sulphate was always suciently available. With the exception of phosphate, all the macronutrients determined
were available until the end of the batch, when the C
source was depleted as well. The initial rapid depletion
of PO3
4 may indicate a relling of an internal phosphate
reservoir (e.g. phytin), because further growth after
PO3
4 depletion was obtained. The pH was between 4.0

and 4.8 during culture, although the fresh medium was

adjusted to pH 5.6.
The medium in which the A. hippocastanum suspension culture was started contained the amino acids glutamine, serine, and glycine. These amino acids showed
rapidly decreasing concentrations after inoculation and
medium exchange. Even after total depletion, the further
growth was not signicantly inhibited (Fig. 5c). Therefore, amino acids were not essential for A. hippocastanum cell culture. Other amino acids (Asp, Glu, Asn, Ala,
Tyr) sometimes occurred during a fermentation (data
not shown) and it was uncertain, whether they were secreted by viable cells or set free after lysis of dead cells.

Discussion
In this paper a bench-scale reactor system for plant cell
cultivation was introduced. It met the requirement for a
high-density suspension culture with the advantages of
bubble-free aeration. A specially designed membrane

154

stirrer allowed gentle agitation, without being clogged

by cell aggregates. It was shown that the oxygen transfer
capacity was sucient for high-cell-density A. hippocastanum culture. This reactor design represents a culture
system that allows the control of the culture parameters
(temperature, dissolved oxygen, pH) under homogeneous conditions. As the scaling-up is limited, it may be
more suitable for physiological investigation, e.g. of
secondary metabolite formation, than for large-scale
biomass production.
Acknowledgement The authors wish to thank H. Buntemeyer and
A. Stenner for HPLC measurements.

References
Buntemeyer H, Lutkemeyer D, Lehmann J (1993) Optimization of
serum-free fermentation process for antibody production.
Cytotechnology 5: 5767

Heidemann R, Riese U, Lutkemeyer D, Buntemeyer H, Lehmann J

(1994) The SuperSpinner. A low cost animal cell culture bioreactor for the CO2 incubator. Cytotechnology 14: 19
Lehmann J, Piehl GW, Schulz R (1987) Bubble free cell culture
aeration with porous moving membrane. Dev Biol Stand 66:
227240
Murashige T, Skoog F (1962) A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol Plant 15:
473479
Piehl GW, Berlin J, Mollenschott C, Lehmann J (1988) Growth
and alkaloid production of Thalictrum rugosum in shake asks
and membrane-stirrer reactors with bubble free aeration. Appl
Microbiol Biotechnol 29: 456461
Schmitz U, Lorz H (1988) Changes in anion and sugar content in
liquid media during in vitro culture of cells from cereals, sugarcane and tobacco. In: Pais MSS, Mavituna F, Novais JM
(eds). Plant cell biotechnology. NATO ASI Series, vol H18.
Springer, Berlin Heidelberg, pp 291296
Wang DIC, Cooney CL, Demain AL, Dunnil P, Humphrey AE,
Lilly MD (1979) Fermentation and enzyme technology, Wiley,
New York, pp 173175