Professional Documents
Culture Documents
Springer-Verlag 1997
ORIGINAL PAPER
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
150
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
Inoculum
N2
CO2
pO2
pH
Temperature control
Magnetic stirrer
Medium
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 in
O2
Sample port
55 mm
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.
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.
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
152
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
qO2 (mg g
dry wt)1 h)1)
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
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
153
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
References
Buntemeyer H, Lutkemeyer D, Lehmann J (1993) Optimization of
serum-free fermentation process for antibody production.
Cytotechnology 5: 5767