Multiphase Bioreactor Design

15–20 g DW L−^1 (Hashimoto and Azechi, 1988; Ritterhaus et al., 1990; Hibino and
Ushiyama, 1999).
In most practical applications, it is a secondary metabolite which is of interest and not
the biomass itself. Achievable levels of important metabolites have been enhanced
through a combination of line selection and processing strategies (Buitelaar and Tramper,
1992) and novel approaches offer potential for further improvements (Shuler, 1999;
Verpoorte et al., 1999). Levels for commercially significant and/or high-yielding cell
lines are summarised in a number of recent papers (Sahai, 1994; DiCosmo and Misawa,
1995; Curtis, 1999). The highest level achieved to date (36% of DW) is for rosmarinic
acid in suspensions of by Salvia officinalis (Hippolyte et al., 1992). Extracellular
paclitaxel levels of up to 117 mg L−^1 have been detected in Taxus canadensis cultures
(Ketchum et al., 1999); Bringi et al. (1997) reported whole-broth paclitaxel titres of 902
mg L−^1 in fed-batch cultures of Taxus spp.. In general, secondary metabolite
productivities are typically of the order of 10−^2 − 10 −^1 g L−^1 d−^1 (Scragg, 1995). From a
processing perspective, it is important to note that many plant cells retain the metabolite
of interest intracellularly, which complicates product recovery and which has led to an
interest in the development of non-lethal methods for inducing product release. Growth-
associated ECPs, which accumulate in the suspending medium, are an important
exception. Use of a perfusion system for the high density cultivation of A. officinalis
yielded a 2.4-fold increase in extracellular protein levels, compared to a batch system (Su
et al., 1996) and offers potential for the production of heterologous proteins.
Most plant cell suspensions can be cultivated at temperatures of between 15 and 35°C,
with temperatures of 25–28°C most common. Limited studies of the effects of
temperature on growth and product formation by C. roseus (Fowler, 1988) indicated
maximum serpentine productivity at 25°C, with maximum growth rates observed at
30°C. However, this phenomenon may reflect the decoupling of biomass and secondary
metabolite formation for non-or partially growth-associated product formation, rather
than a temperature-specific effect.

BIOREACTOR DESIGN AND OPERATION

Mixing and Mass Transfer

In any bioreactor, the purpose of mixing is to ensure homogeneity with respect to
biomass and nutrients, which includes effective gas phase dispersion. Plant cell broths are
characterised by lower oxygen and cooling requirements (Section on Oxygen
Requirements) than microbial fermentation fluids, but typically have higher apparent
viscosities (Section on Broth Rheology). Reducing the intensity of the hydrodynamic
environment to which potentially shear sensitive systems (Section on Shear Sensitivity)
are exposed, may conflict with the need to prevent particle settling in heavily aggregated
suspensions, with a solid specific gravity of approximately 1.02–1.03. Although particle
sedimentation is generally not a problem in finely dispersed suspensions, increased
aggregate size, which may be conducive to secondary metabolism and which, moreover,
has been reported to occur on scale-up, leads to an increase in the minimum impeller
speed (Njs) required for particle suspension. Using the Zwietering (1958) correlation to

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