The shaded regions for the Atropa belladonna data reflect the displacement of the data
depending on whether total cumulative energy dissipated in the broth is considered, or
that fraction to which the biomass alone is exposed (at a maximum level of 13 g DW L−^1 ,
which corresponds to an estimated 26% volume fraction, assuming a representative
FW/DW ratio of 20). Overall, there is a good level of agreement between the response
trends. The results suggest that cell metabolism, represented in this case by mitochondrial
activity (curve (c)), may be affected at cumulative energy dissipation levels significantly
below those required to cause detectable damage to the cell membrane (curve (a)). Doran
(1999) used the A. belladonna data to identify a threshold level of 10^7 J m−^3 for shear-
related damage in stirred bioreactors. Although undeniably valuable as a correlating
parameter for cell response, the use of cumulative energy dissipation does not elucidate a
mechanism for cell damage.
The current consensus on shear sensitivity is that plant cells are considerably more
robust than was initially believed. Nonetheless, reduction in overall productivity on
scaleup is common and, where cell death is not a factor, it reflects sub-lethal system
responses. However, a full understanding of the key processes in stress perception and
stress responses in plant cells has yet to be achieved. Identification of appropriate
cultivation conditions can be approached using the guidelines presented in this section,
but it is still a non-trivial, line specific task.
Performance in Suspension CultureIn suspension culture, plant cells behave in a generally similar fashion to microbial
systems. The most significant difference is the duration of batch processes. Typical
doubling times for plant cells of between 1 and 4 days result in batch cultivation periods
of between 5 and 25 days. Inoculum volumes are typically 5–15% of the total broth
volume. Particularly low apparent growth rates may be attributable to appreciable levels
of cell death and lysis, caused by sub-optimal cultivation conditions. For M. sativa L.
suspensions in a 10 L STR, cell viability was observed to fall from 80% to 50%, over the
first 3 days of cultivation, independent of nutrient limitation (Steward et al., 1999b).
Furthermore, from day 7 onwards, cell lysis accounted for more than 20% of total cell
death. Accordingly, calculation of apparent specific growth rates, on the basis of whole
cell counts alone, significantly underestimated actual rates of cell proliferation. Extended
fermentation times challenge system sterility and make plant suspension processes
inherently less commercially attractive, unless they have a competitive edge over existing
or potential alternatives for the production of the same material.
Maximum biomass levels in the range 10–20 g DW Lr−^1 can now be routinely
achieved with most cell lines. Biomass yields are approximately 0.5–0.6 g DW (g
sucrose)−^1 ; while a value of 0.78 g g−^1 has been reported (Pareilleux and Vinas, 1983), it
is likely that this reflects conversion of the carbon source to intracellular stored
carbohydrates, rather than to biomass. As with microbial systems, biomass levels can be
augmented by optimisation of cultivation conditions, including substrate feeding and
medium perfusion, and concentrations of 70 g DW L−^1 (Fujita, 1988b) have been
reported. However, from the comparatively limited data available, it appears as if
biomass levels for commercial and commercial-scale processes are typically in the range
Bioreactor design for plant cell suspension cultures 433