Liquid Culture Systems for in vitro Plant Propagation

Development and validation of low cost bioreactor 515
3. Results and discussion
3.1 Control culture in 250 ml Erlenmeyer flasks
At first, shoot growth (Figure 3) was characterized in 250 ml Erlenmeyer
flasks (Figure 12 A). From a 5 g FW inoculum, after a lag period of 3 days,
linear growth followed, reaching a final biomass fresh weight of 18 g after
23 days. As a function of time, sucrose is rapidly hydrolysed and glucose
and fructose progressively consumed. In such conditions, the total FCs level
is typically of 1.2 % DW (Massot et al., 2000). Shoot growth occurred at a
mean rate of 0,06 g DW per day, corresponding to 1.36 mg of FCs produced
per day of culture and per gram DW of inoculum over 23 days (6.8 mg FCs
day -1 l -1 ).
3.2 Establishment of a reference bioreactor culture
Ruta shoot culture was performed in a 4-litre SGI bioreactor coupled to a
5 litre Schott (Bioreactor 1: Figure 2A and Figure 12 B), the whole
containing 5.8 litres of B5(30)K2D2 medium with a 115 g FW inoculum. As
presented in figure 4, growth occurred with a maximum rate of 13 g FW day -1
for 16 days and thereafter, only 2 g of fresh weight were produced each day
for the further 11 days of the experiment. Starting from 115 g FW, the total
biomass reached 332 g FW within 27 days. Sucrose was completely
hydrolysed in 6-7 days whereas glucose and fructose were progressively
consumed during the culture (data not shown). At day 27, only 1 g of
glucose and fructose still remained in the culture medium. The
furanocoumarin concentration in the medium oscillated between 1 and
6 mg l -1 and the relative proportion of the four furanocoumarins is relatively
constant during the culture period. Shoot growth occurred at a mean rate of
0.8 g DW per day corresponding to a calculated FC production of 0.98 mg of
FCs per day of culture and per gram DW of inoculum in 4.8 l of medium
(2 mg FCs day -1 g -1 l -1 ). The productivity per volume unit is three times
lower than the results obtained with the Erlenmeyer flask cultures, but the
total daily FC production is much higher (9.78 vs 0.68 mg FC per day).
In this bioreactor system, the biomass spatial distribution exhibited
notable heterogeneity. Indeed, the shoots were rapidly pinned between
bioreactor glass wall and vertical parts (tubes and probes). This
heterogeneity of shoot distribution in this bioreactor led us to think that it
could be, in the future, a source of variability in terms of growth and FC
production. Furthermore, the biomass cannot be inoculated via the tubing of
the reactor, meaning that a further scaling up is hardly conceivable.