Liquid Culture Systems for in vitro Plant Propagation

Bioreactor design for propagation 47
This construction was first described at the WG2 meeting of COST 87 in
Aas (Hvoslef-Eide and Heyerdahl, 1992).
The gasses cross the silicone tube walls at a rate given by the partial
pressure difference across the wall, the net direction being from the high to
the low partial pressure side (Luttmann et al., 1993). In addition to supplying
oxygen to the medium, the tubes therefore also remove gasses produced by
the cells, e.g. carbon dioxide and ethylene, which have a higher partial
pressure in the medium than at the air/gas tube inlet.
Measuring the pressure, and then manipulating the mixing ratio and flow
rate of air and gas through the gas exchange tube controls the oxygen partial
pressure of the medium. Besides obtaining a correct oxygen concentration
according to the set point, the controller must also maintain a sufficient flow
through the tubes to constantly remove gasses produced by the cells.
A gas mixer designed for these bioreactors controls the flow rate of air
and additional gases. The gas mixer is two independently controlled flow
regulators, one for air and one for the enrichment gas. Each flow regulator
consists of a solid-state gas flow sensor (Honeywell AWM 3100V), an
analogue PI controller and a gas tubing pinch valve actuated by a bidirectional-controlled
motor. An eccentric cam (disk) on the motor shaft,
pinches the gas tubing more or less, thus effecting the valve opening and
closing. The control computer sets the percent flow set point for this control
loop, and the motors are actuated until the correct gas flow is achieved. The
gases from the two flow controllers are then mixed in a tube prior to the
bioreactor gas inlet port.
This method functions well for oxygen concentrations ranging from 50-
150% when the suspension has a medium amount of cells that use oxygen.
For lower oxygen concentrations, or in cases where the cell number is so
small that the cells do not use enough to keep the concentrations low, we
have to use nitrogen in the gas mixture to be able to maintain such low set
points. On the other hand, when the cell count is high and the suspension is
thickening, it is impossible to keep high set points even when supplying pure
oxygen. These limitations must be monitored closely during the
experiments.
2.4 pH measurement and control
We have used the pH electrodes (Ingold 405-DPAS-K8S/200
combination pH electrode and Mettler Toledo 405-DPAS-SC-K8S/200, pH
0-12, 0-130 o C). There can be a considerable calibration drift in a pH
electrode during autoclaving and in the course of an experiment (our
unpublished results). The pH electrode is calibrated before each experiment
by standard solutions at pH 4 and 7. The slope of the electrode calibration is