Liquid Culture Systems for in vitro Plant Propagation

46 Anne Kathrine Hvoslef-Eide et al.
2.3 Oxygen measurement and control
The oxygen electrode (Ingold InPro 6100/220/T/N) measures the partial
pressure of dissolved oxygen molecules. This is the most expensive single
component of the bioreactor. The electrode must be of a type that can stand
repeated autoclaving and need to be accurate and not drift too much during
the course of an experiment of up to 8-10 weeks. Since the electrode
membrane must be in contact with the growth medium, the electrode cannot
be removed for calibration after sterilisation has taken place. The electrode is
therefore calibrated in situ after sterilisation but before inoculation. The first
calibration step is to allow laboratory air of approximately 21 % oxygen to
flow through the bioreactor gas exchange tube for 24h at the set temperature.
The measured value in this condition is then defined to be 100 % partial
oxygen pressure (Preil, 1991). Such oxygen electrodes have an insignificant
current at zero oxygen pressure. We therefore disconnect the oxygen
electrode to simulate the zero point. This value thus represents the zero point
error of the electrode amplifier, and this value is defined as the 0 % oxygen
partial pressure point. Oxygen electrodes have linear characteristics and
because two points (0 and 100%) have been defined, a linear calibration
curve for the oxygen measurement of that particular experiment can be
drawn. We can provide gas sparging between 0 and 150 % oxygen in the
bioreactors. Zero can be provided by using pure nitrogen, while 150 % can
be obtained through enrichment with pure oxygen.
Oxygen is provided bubble-free by using silicone tubing (Figure 1B)
with the following specifications: inside diameter 3 mm, wall thickness 0,4
mm (H.Jürgens and Co, Bremen, Germany, No.9.205 256), similar to those
used by Preil et al. (1988). This tubing allows gas exchange, but the pores
are small enough to prevent bacteria and fungi going through, thereby
providing a sterile barrier. We therefore do not use sterile
air/oxygen/nitrogen, but laboratory air driven through the silicone tubes by
an aquarium pump (Rena 301, 6 W, 600 l h -1 , Rena, Annecy, France) The air
is enriched with pure oxygen or nitrogen as needed. This gas mixture enters
through an inlet in the bioreactor lid, flows through the metal and silicone
tubes and eventually is exhausted via the outlet.
The air tube consists of 20 silicone tubes, each 12 cm, connected by Ushaped
stainless steel pipes welded to the lid (Figure 1B). Similar U-shaped
stainless pipes are used to connect the short lengths of silicone tubing,
additionally providing some ‘weight’ to assist the tubing to hang downwards
into the medium. By this arrangement the tube loops hang loosely under the
lid constantly dancing in the medium eddies. This is in contrast to designs
with stationary tubes where cells might grow on the outside of the tubes.