Liquid Culture Systems for in vitro Plant Propagation

Bioreactor design for propagation 49
The pH can be controlled (very) precisely, but one problem then arises:
acid and alkali are then more or less constantly added to maintain the set
point. This produces salt that may affect the biological processes in the
bioreactor. To avoid unnecessary elevation of salt concentration, our
controller implements a dead band around the set point. As long as the pH is
within the dead band, the controller will not activate the alkali or acid
pumps. We have not used active pH regulation for our experiments in many
cases, as the cells seem to be growing and developing well at the pH range
provided. Therefore, we have avoided pH regulation to simplify
experimental design as much as possible. But the pH of the medium is a
good indicator for the condition of a cell suspension, and any infection is
usually first spotted as an abnormal pH reading. Normally, with the medium
we use, the pH reduces after inoculation, and then rises again (Figure 3).
2.5 Stirring speed and stirrer direction
The stirrer is a pitch blade stirrer made of stainless steel (Figure 1B and
1F). The shaft is held by a ball bearing pressed into a hole in the bioreactor
lid. The bearing provides enough stability for the stirrer. The grease of this
bearing can stand the autoclave temperature well and takes part in the sterile
barrier. The motor and stirrer shaft are coupled by a slot and tap to allow for
easy removal of the motor when the vessel is taken away. The rotational
speed can be varied from 0 - 100 rpm. The regular changes of direction are
important to obtain a good mixing at low speeds and hence reduce the shear
forces. A speed as low as 30 rpm is sufficient to provide good mixing when
changing the direction every 10s.
2.6 Light quality provision
It is possible to expose the cultures to different light qualities by using a
light source providing near daylight spectrum (OFT bioLIGHTSYSTEMS,
Strand Lighting, UK). This light can then be filtered through different
coloured stagelight filters that will filter away parts of the light spectrum.
We have used a blue filter (Strand filter No 419, primary dark blue) and a
red filter (Strand filter No 406, primary red). All light treatments were at the
same light quantity; 5 µmol m -1 s -2 for 18 h. Measuring the light quantity
inside the bioreactors and moving the light source closer or further away to
provide the same quantum after filtering can obtain this. The bioreactors can
be covered with aluminium foil to provide darkness.