suspension culture, cultivated in shake
flasks (Bar represents 50 μm).
(a) Morinda ctirifolia and (b) Drosera capensis cells, cultivated in 250 mL shake flasks.
M. citrifolia cells are characteristically cylindrical in shape and exist in the form of
unbranched chains of between 2 and 15 cells. D. capensis grows in the form of
aggregates which continuously slough off smaller aggregates and single cells into the
suspending medium. Each plant cell is surrounded by a cell wall which defines cell size
and shape and which plays a dynamic role in cellular expansion and osmo-regulation.
Unlike microbial cells, a significant portion of the cellular volume is occupied by a
vacuole, into which secondary metabolites are frequently sequestered and which is
generally observed to increase in size during batch cultivation. With a view to ensuring
system homogeneity and minimising mass transfer limitations, suspensions of single cells
appear most desirable. However, aggregates, consisting of between 2 and many hundreds
of cells, are most common. The predominance of aggregates is often associated with the
presence of extracellular polysaccharides (ECPs), which may retard cell-cell separation
after division. Cell and aggregate morphology vary with cell line, growth stage and
cultivation conditions. For example, Curtis and Emery (1993) reported a switch between
elongated and spherical cells in suspensions of Nicotiana tabacum cultivated under batch
and semi-continuous conditions, respectively. Aggregation patterns are also known to be
strongly influenced by hydrodynamic environment; transfer from shake flask to stirred
tank reactor (STR) has been reported to both increase (e.g. Wagner and Vogelmann,
1977) and reduce (Takeda et al., 1994) average aggregate size. In laboratory-scale
bioreactors, sampling ports/devices designed for microbial broths are frequently
unsuitable for aggregated plant suspensions and wider bores should be employed.
Cell-cell contact is generally regarded as advantageous for secondary metabolism and
aggregate size-related variations in metabolite productivity have been attributed to a
number of factors. Some degree of cellular organisation or differentiation is thought to be
beneficial and is reflected in the heightened productivity reported for many tissue and
organ cultures. Enhanced secondary metabolite production has been observed in compact
aggregates of self-immobilised cells of Solanum aviculare (Tsoulpha and Doran, 1991)
and Rhodolia sachalinensis (Xu et al., 1998). The Nitto Denko ginseng process employs
a highly aggregated Panax ginseng suspension (Hibino and Ushiyama, 1999) with
aggregate equivalent diameters of up to 2 cm (Hibino, 1999). In this case, productivity is
extremely sensitive to aggregate disruption and agitation rate has been identified as the
most significant factor for secondary metabolite production. Diffusional limitations may
prevail in larger aggregates and Hulst et al. (1989) estimated a critical diameter of
approximately 3 mm for oxygen limitation, at a bulk oxygen concentration of 0.25 mmol
L−^1. At lower broth DO levels, the critical aggregate diameter will be reduced. However,
even for aggregates of this size, or smaller, nutrient availability can be significantly
influenced by the prevailing flow patterns: Singh and Curtis (1994a) observed hollow
aggregates in an air-lift reactor (ALR), while solid aggregates of a comparable size were
formed in the more turbulent flow field of a STR. Variations in light intensity may also
occur throughout aggregates (Madhusudhan and Ravishankar, 1996), which will impact
on photoautotrophic cultures and on systems in which metabolite production (e.g.
anthocyanins) is stimulated or enhanced by light.
Multiphase bioreactor design 422