Biochemical Engineering Journal 4 (2000) 8999

Bioreactors for plant engineering: an outlook for further research

Lidija Sajc a , Dragan Grubisic b , Gordana Vunjak-Novakovic c,
a

Department of Analytical Chemistry, Faculty of Technology, Belgrade University, Karnegijeva 4, PO Box 494, 11000 Belgrade, Yugoslavia
b Department of Plant Physiology, Faculty of Biology, Belgrade University, 29 novembra 142, 11000 Belgrade, Yugoslavia
c Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Received 1 September 1998; accepted 21 May 1999

Abstract
This paper reviews bioreactor related aspects of large scale plant cell technology for the production of biologically active compounds.
Bioreactor designs currently in use are discussed with respect to specific operating parameters that can be varied to modulate cell growth
and function in order to optimize product release and separation. Flow and mixing are recognized as key factors responsible for both
the direct hydrodynamic effects on cell shape and function and flow induced changes in mass transfer of nutrients and metabolites. The
integration of biosynthesis and separation is considered as a possible approach towards more efficient plant cell and tissue culture. 2000
Elsevier Science S.A. All rights reserved.
Keywords: Plant cell bioreactors; Plant cell culture; Mixing; Bioseparations

1. Introduction

2. Approaches to plant tissue bioprocess engineering

The large scale plant cell and tissue cultures have been
considered as an alternative source of biochemicals over the
last 40 years. Routien and Nickel received the first patent
for the cultivation of plant tissue in 1956 [1] and suggested
its potential for the production of secondary metabolites
[2]. Shortly after that time, the National Aeronautics and
Space Administration (NASA) started to support research
of plant cell cultures for regenerative life support systems.
Since early 1960s, experiments with plants and plant tissue
cultures were performed under various conditions of microgravity in space (one-way spaceships, biosatellites, space
shuttles and parabolic flights, the orbital stations Salyut and
Mir) and accompanied by ground studies using rotating clinostat vessels [3]. Growth, development and metabolism of
plant cells and tissues have been studied to improve our understanding of plant cell biology and tissue physiology, and
derive criteria for bioprocess design [4].

The term Plant Tissue Culture in this paper refers to the

in vitro cultivation of any plant segment, whether a single
cell, a tissue or an organ [5]. There are five main types of
plant tissue cultures: (1) seedlings of plants (plant cultures),
(2) isolated embryos (embryo cultures), (3) isolated plant
organs (organ cultures), (4) explant cultures (tissue or callus
cultures), and (5) isolated cells or small aggregates dispersed
in liquid media (cell or suspension cultures).
The concept of using plant cell cultures is confined to
the production of valuable natural products such as pharmaceuticals, flavors and fragrances, and fine chemicals over
20 000 different chemicals are produced from plants, with
about 1600 new plant chemicals added each year. According
to an OECD report [6,7], plant derived drugs and intermediates account for approximately 911 billion US$ annually
in the US alone.
Generally, the plant products of commercial interest are
secondary metabolites, which in turn belong to three main
categories: essential oils, glycosides and alkaloids. The essential oils consist of mixture of terpenoids, which are used
as flavoring agents, perfumes and solvents. The glycosides
include flavanoids, saponins, phenolics, tannins, cyanogenic
glycosides and mustard oils, which are utilized as dyes, food
colors and medicinals (e.g., steroid hormones, antibiotics,
digitalis). The alkaloids are a diverse group of compounds

Corresponding author. Tel.: +1-617-253-3858; fax: +1-617-258-8827

E-mail address: gordana@mit.edu (G. Vunjak-Novakovic)

1369-703X/00/$ see front matter 2000 Elsevier Science S.A. All rights reserved.
PII: S 1 3 6 9 - 7 0 3 X ( 9 9 ) 0 0 0 3 5 - 2

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with over 4000 structures known; almost all naturally occurring alkaloids are of plant origin. Alkaloids are physiologically active in humans (e.g., cocaine, nicotine, morphine,
strychnine) and therefore of a great interest for pharmaceutical industry [8].
Secondary metabolites are currently being obtained commercially by extraction from whole plants. Large scale plant
tissue culture is an attractive alternative to the traditional
methods of plantation, as it offers two advantages: (1) controlled supply of biochemicals independent of plant availability (climate, pests, politics), and (2) well defined production systems which result in higher yields and more consistent quality of the product [9].
However, various problems associated with low cell productivity, slow growth, genetic instability of high-producing
cell lines, poor control of cellular differentiation and inability to maintain photoautotrophic growth have limited the application of plant cell cultures. Also, much of the research
in this field has been done by scientists associated with private industry, not published and not available to academic
institutions [10]. In addition, plant cell culture is expensive, and the best product candidates are those of high value
(5001000 US$ per kg) and low-volume, not synthesized
by microorganisms, and too complex for chemical synthesis
to be a reasonable alternative. As a rule of thumb, the process is commercially feasible if it yields a revenue of about
12 c/l/day [11].
In spite of potential advantages of the production of secondary metabolites in plant cell cultures, only shikonin, ginsenosides and berberine are presently produced on a large
scale, and all three process plants are located in Japan [12].
In addition, the anti-cancer drug Taxol (registered trademark of Bristol-Myers Squibb) is currently under consideration for large-scale production [1315]. Consequently,
the ongoing research in plant cell culture is largely focused
on the identification of rate limiting steps in biosynthetic
pathways. Several approaches were investigated: elicitation
followed by monitoring the activities of pathway enzymes;
measuring enzyme levels in cell lines of different biosynthetic capacities or after an addition of precursors; and determining the transformation and over-expression of pathway
genes [13,1621]. However, the design of biochemical reaction networks requires identification of the critical branch
point(s) and determination of the exact enzyme(s) that must
be modified. Although molecular biology has provided the
means to conduct precise genetic changes, the difficulties
with targeting specific enzymes are still the limiting factor
of faster progress in this area [22].
Advances in molecular biology hold promise for new
products and new processes in plant biotechnology. Two
major strategies for creating transgenic plants have been
devised: (1) genetic transformation of the plants using
Agrobacterium gene vectors or direct gene transfer by
protoplast fusion, microprojectile bombardment, or electroporation, and (2) genome manipulation of plant pathogenic
viruses. Genome manipulation is resulting in relatively

large amounts of desired compound produced by a plant

infected with an engineered virus, whereas transgenic
plants can maintain constant levels of production of proteins without additional intervention. Genetic manipulation of plant cells poses several fundamental problems,
including the stability of inserted genes, the functional
expression of the enzymes involved, the localization of
enzymes and products within the cellular compartments,
and the effects of an additional pathway on the growth
and physiology of plant cells. As a result, the productivity
of only a few medicinal plants has been increased in this
way [2327]. Plant cell cultures can be designed to produce valuable therapeutic proteins, including monoclonal
antibodies, antigenic proteins that act as immunogenes
(edible subunit vaccines for rabies, cholera, hepatitis B,
malaria), human serum albumin, interferon, human enzyme
for treating Gauchers disease, immuno-contraceptive protein, ribosome inactivator trichosantin, antihypersensitive
drug angiotensin, leu-enkephalin neuropeptide, and human
hemoglobin [20,2834].
Due to an increased appeal of natural products for medicinal purposes, the metabolic engineering can affect the
production of pharmaceuticals and help design new therapies. The candidate plant cell cultures are generally chosen
by screening from medicinal and aromatic plants already
used in drug production. At present, research and development are focused on plants producing substances with
immunomodulating, antiviral, antimicrobial, antiparasite,
antitumor, anti-inflammatory, hypoglycemic, tranquilizer
and antifeedant activity [35]. The delivery of these drugs
is often more complicated than that of conventional drugs,
necessitating gene therapies or controlled drug release systems based on biodegradable polymer materials [3639].
Advances in material science, reactor design and integration of the existing and novel separation processes into an
overall processing scheme can further improve large scale
plant tissue engineering.

3. Process design considerations

Plant biotechnology requires a cross-disciplinary research
in plant physiology, cell and molecular biology, pharmacology, toxicology, chemistry, and chemical engineering to assess: (1) tissue morphology and composition; (2) flow and
mass transfer conditions in the bioreactor; (3) kinetics of
cell growth and product formation; (4) genetic stability of
high-producing cell lines; (5) control of bioreactor micro
and macro environment, (6) implications of bioreactor design on downstream processes; and (7) the potential for process scale up.
The goals of plant tissue process development are to
achieve high productivity (g product/l/day), high product
yield (g product/g substrate) and high product concentration (g product/l) by selecting cell lines, media and bioreactor operating conditions. A combination of various prod-

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uct enhancement strategies, including the in situ product removal by extractive plant cell culture, the use of cell-polymer
constructs, application of different precursors, elicitors, and
medium exchange strategies, can improve the plant tissue
culture more than any of the above factors alone.

4. Plant tissue constructs

Immobilization of plant cells on or within polymer matrices generally affects plant cell differentiation and often results in higher productivity when compared to a cell suspension. Several methods have been devised for plant cell immobilization: (1) attachment on carrier beads, nets or foam
surfaces, (2) entrapment into porous gel beads made of natural (alginate, agarose, k-carageenan, gelatin, chitosan) or
synthetic polymers and copolymers, and (3) entrapment by
membrane barriers in flat sheet or hollow fiber membranes or
microcapsules. The above procedures have been extensively
studied with respect to cell viability and function, physical
and mass transfer properties of the immobilization support
[4044].
Plant tissue constructs can offer significant advantages
over cell suspensions: (1) high cell concentration which improves cellcell communication and increases the volumetric
productivity, (2) easy exchange of medium, which maintains
Newtonian flow properties, (3) shear-protection of the cells,
(4) continuous removal of products and inhibitory metabolites which can enhance cellular metabolism or unmask biochemical pathways, (5) better control of cell differentiation
and the production of secondary metabolites, and (6) advanced separation methods (e.g. magnetic separations based
on the incorporation of magnetite, Fe3 O4 , into the immobilization matrix).
Aggregation of plant cells in suspension is common,
and the aggregation phenomena has been studied as
self-immobilization method [4546]. Aggregation patterns,
studied by image analysis [47] and sieving [48] can vary
with the cell line, culture age, and cultivation conditions.
Wagner and Vogelmann [49] observed that a scale-up from
shake flask to bioreactor cultivation was accompanied with
a change from pellet culture to single cell suspension, which
emphasized the importance of assessing cell and tissue
morphology for actual environmental conditions and culture
age. Shuler [50] also suggested that metabolite productivity
may be significantly influenced by the degree of cellular
association and may therefore be affected by variations in
aggregation patterns during scale up.
The morphology of suspended cells and aggregates
can significantly affect the apparent viscosity of suspension. Relevant studies, using different types of viscometers show that the majority of cell suspensions exhibit
time-independent non-Newtonian (Bingham plastic, pseudoplastic) shear-thinning rheology [47,5153]. Thixotropic
behavior, as one example of time-dependent change in shear
stress was observed by Wagner and Vogelmann [49].

91

5. Aeration
Most nutrients required for cellular growth and metabolism
are highly soluble in water and sufficient amounts can be
initially incorporated in the medium to support the slow
metabolism of plant cells for extended periods of time.
Oxygen, as the major exception, needs to be supplied
throughout the cultivation. Since the volumetric productivity of high-density and high-viscosity cell suspensions
is generally proportional to cell concentration, which is in
turn limited by oxygen supply, aeration is a major concern
in bioreactor design and scale up for plant cell culture. In
principle, the rate of gas exchange has to be determined
to provide sufficient supply of oxygen while preventing
excessive removal of CO2 and essential volatiles from the
system. Studies of gas exchange in plant suspension cultures have focused on the overall oxygen mass transfer
coefficient, and its dependence on the rates of aeration and
liquid mixing. Oxygen requirements of plant cells are relatively low for cell growth, but may significantly increase
during metabolite synthesis [54].
6. Mixing
The other key parameter is mixing, which is necessary
to equally distribute cells, nutrients and products through
the liquid phase. Mixing is normally carried out by sparging, mechanical agitation, or a combination of these two,
to maintain uniform concentration of chemical species (e.g.
pH, gases and nutrients) in the bulk phase and to increase
the rates of mass transfer.
In vitro, morphogenesis of engineered tissues is expected
to depend on exogenous factors, i.e., flow and mixing, in at
least two ways: (1) by direct hydrodynamic effect on cell
shape and function, and (2) by flow-induced changes in mass
transfer rates of nutrients and metabolites. However, there
has been little quantitative work on the effect of hydrodynamic forces on plant cells that are relevant for bioreactor
design and operation for plant tissue engineering. Previous
studies have focused mostly on the kinetics of cell growth
and product formation, and the effects of hydrodynamic conditions on the structure and composition of plant tissues are
not well understood.
The magnitudes of hydrodynamic forces associated with
mixing should be low enough not to cause cell death, but
sufficient to stimulate selected cell functions. In well mixed
bioreactors, hydrodynamic effects are caused by the time
and space fluctuations in liquid velocity and pressure that are
associated with viscous dissipation of turbulent eddies. The
basic structure of turbulence involves spectrum of eddy sizes,
where larger eddies pass their kinetic energy to smaller ones.
The smallest, Kolmogoroff size eddies, release their kinetic
energy by viscous dissipation, which is the key mechanism
by which turbulence affects cell shape and function.
In order for the cells not to be damaged by turbulence, the
smallest eddies should be large when compared to the cells,

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and still much smaller than the whole tissue sample (or immobilized cell carrier). Due to the lack of mobility, immobilized cells are exposed to pressure and velocity fluctuations
which may stimulate cell metabolism and affect cellcell
interactions. However, when the dimensions of the smallest
turbulent eddies containing significant energy approach the
dimensions of a cell, gradients in shear stress are established
over distances comparable to cellular dimensions, and the
cell is exposed to a twisting motion. It has been well documented that the cell is considerably more sensitive to the
fluctuating turbulent as compared to steady laminar shear
[55,56].
Shear studies of plant cells and tissues can be divided
into two categories: (1) the cells were exposed to shear
forces under growth conditions for the entire duration of
cultivation, and (2) the cells were exposed to well defined
shear forces (e.g. laminar or turbulent flow in Couette, capillary and submerged jet devices) for short periods of time,
generally under non-growth conditions. Reduction in cell
viability (growth rate, regrowth potential, membrane integrity dye exclusion, dual isotope labeling and dielectric
permitivity experiments), release of intracellular components (variation in pH, release of proteins and secondary
metabolites), change in metabolism (oxygen uptake rate,
ATP concentration, metabolite synthesis, cell wall composition), and changes in cell morphology and aggregation
patterns were among shear-related effects identified in plant
cell cultures [5759]. Recent studies also involved calcium
transport, expression of stress proteins and cytoskeletal
changes as shear stress indicators [60].
Many authors used the concept of a critical shear stress,
above which cell viability is lost. For laminar flow conditions, Vogelmann et al. [61] suggested a critical shear
stress between 80 and 200 N/m2 . For cell suspensions,
shear-related damage was correlated to the dissipation of
energy [6264]. The values reported for plant cells are generally much higher than those reported for mammalian cells
[65], which could be explained by the presence of a thick,
rigid, cellulose-based cell wall.
The levels of shear stress in shake flasks and airlift bioreactors, can be estimated as follows based on the reported data
[6668]. The smallest eddies in shake flasks (417 mm) and
in airlift bioreactors (71 mm) were large when compared to
cell size (30 mm), but much smaller than the construct size
(2.5 mm). Turbulent shear stress of 0.01 N/m2 in shake flasks
was much lower than the value of 0.15 N/m2 , reported by
Davies et al. [55] to cause mammalian cells turnover. However, this relatively low level of turbulent shear, if applied
over a long time of cultivation (typically 4 weeks), stimulated the cells. According to Stathopouloulus and Hellums
[68], laminar shear stresses below 0.26 N/m2 have no effect on mammalian cell viability, but may induce cell alignment in the direction of flow; intermediate stress levels over
0.65 N/m2 cause further morphological changes, increase
cell permeability and cause a 1020% loss in cell viability;
stresses above 2.6 N/m2 are detrimental.

Mechanical stress applied to cell surface receptors play a

critical role in the development and maintenance of tissue
patterns. All cells are prestressed structures which generate mechanical force within their cytoskeleton, many times
greater than large-scale forces (e.g. gravity), and exert
tension on their extracellular matrix. Specifically, the cytoskeletons isotropic structural continuum provides an internal 3D-structure which maintains cell membrane integrity
and facilitates chemical reactions by bringing substrates
and enzymes into close proximity [6970]. Wang and Ingber [71] showed that cytoskeleton stiffening in response
to applied shear stress increased in proportion to the level
of applied stress. Associated changes in cytoskeleton can
modulate chemical signaling, since much of the metabolic
machinery has been shown to be associated with the cell
cytoskeleton.
In plant cells, according to Wagner et al. [72] and Wayne
[73], cell surface receptors also mediate transfer of transmembrane forces and regulate tissue formation. In addition, cells change their force balance and modulate specific
functions when cultured on extracellular matrices. In general, cells express tissue-specific functions and do not grow
when attached to malleable substrates, while low differentiation and high growth were observed for rigid substrates.
Consequently, cells remained quiescent and differentiated
when cultured in malleable calcium alginate which permitted cell retraction and rounding. Kordym [4] has shown that
the acceleration of cell differentiation leads to earlier cell
aging, which is characterized by the increases in the activity of lytic enzymes, respiration rate and metabolite transport. Gontier et al. [74] and Curtis et al. [75] recently explained the additional role of calcium in calcium alginate
immobilization-induced elicitation and increased productivity of plant cells.
Therefore, shear levels which are below levels causing
cell damage while still high enough for efficient mixing
in the liquid phase and dispersion of air are considered as
important criteria of bioreactor design. During the process
scale-up, the goal is to reproduce on a larger scale the conditions found to be optimal on a small scale. With higher
reactor volumes, i.e. longer mixing times, mixing rate can
start to interfere with the process control (as it determines
the time constant of system response) and kinetic rate (as
it determines the transport rates of reactants and products
in biochemical reactions). In most cases, the main problem
of process scale up is to provide the same extent of mixing
on different process scales. This concept is also called the
regime concept and assumes the similarity of flow regimes
between various different scales.

7. Selection of the cultivation method

Reactor operation and the choice between batch, fed-batch
or continuous operation conditions for cell or tissue depend
on the dynamics of the specific culture.

L. Sajc et al. / Biochemical Engineering Journal 4 (2000) 8999

Batch cultivations are characterized by constantly changing environmental conditions, and are capable of producing
metabolites associated with any kinetic pattern. Batch studies are used to determine the conditions for maximum productivity. These conditions can be studied over prolonged
periods of time by using fed-batch operation, e.g. by controlled addition of a limiting nutrient.
Steady-state continuous flow, or chemostat operation, with
a constant withdrawal of culture medium and cells is commonly used for the production of high-volume and low-value
growth-associated products, typically primary metabolites
and biomass. The operation of multiple-staged chemostats
can be suitable when passage through several developmental
stages is a prerequisite for production, but a proper design
of such elaborate reactors is complicated by sudden changes
of plant cell culture characteristics and by a limited understanding of cell physiology.
A continuous flow reactor with cell recycle is a flexible
cultivation system. For a very low cell recycle this system
approaches a chemostat, while for a complete cell recycle
the system approaches immobilized cell culture suitable for
production of non-growth associated products. Continuous
flow with complete cell recycle is often referred to as a
perfusion culture.

8. Process optimization
The design of a bioreactor system is an optimizing procedure of balancing biological and engineering factors to
obtain the required capacity and product quality at minimum production costs. The control of parameters that affect
cell growth and product synthesis is an essential aspect of
bioreactor design. Sterilizable devices must be provided for
on-line measurement of process parameters (temperature,
pH, dissolved oxygen, carbon dioxide, foam, level) and sampling of medium and cells for off-line laboratory tests (cell
concentration and viability, tissue morphology, substrate and
product concentration). Control strategies, which range from
the maintenance of basic parameters at constant levels to
sophisticated computer-aided adaptive control systems can
often determine the overall output of the bioreactor.

9. Modeling
Mathematical models are essential for the understanding
of the effects of various parameters on cell behavior. Bioreactor design for plant tissue culture (number of reactors,
batch or continuous operation) and the choice of a process
control strategy should ideally be based on deterministic
mathematical models. However, only few plant cell systems
were modeled to some extent, and further studies of the regulation of biosynthetic pathways are needed to provide more
insight into the behavior of plant cell cultures. Structured
non-segregated models, which assume lumped biomass sys-

93

tem distribution between two or more compartments, have

been used to describe cell growth and secondary product
formation in plant tissue cultures [67,7684].

10. Bioreactor types

Fundamental studies of bioreactors with plant cells involve three important scientific and practical issues related
to bioreactor design and operation: (1) assessment of cell
growth and product formation; (2) analysis and modeling of
the culture dynamics, including the integration of biosynthesis and product separation; and (3) studies of flow, mixing and mass transfer between the phases, in order to define criteria for bioreactor design and scale up. Experimental research should be combined with theoretical analysis to
develop predictive correlations of biological properties and
transport phenomena in the system. For a given application,
the culture conditions can be optimized with respect to cell
support, medium composition and renewal rate, mass transfer of chemical species, and bioreactor fluid dynamics, in
order to define the conditions that are permissive for or even
designed to promote selected cell functions.
Prenosil and Pedersen [85], Scragg and Fowler [86], Kargi
and Rosenberg [87], Payne et al. [88], Panda et al. [89],
Scragg [2], and Doran [90] reviewed different reactor configurations for plant cell suspensions, plant tissue and organ
cultures. The relative advantages and selection criteria for
various reactor configurations were discussed for specific
process applications. In particular, bioreactors which integrate biosynthesis with product release and separation were
most extensively studied in Japan [91101].
Numerous modifications of the conventional stirring tank
reactor (STR) with bubble aeration have been developed by
employing a variety of impeller designs (Fig. 1Aa). The controllability and flexibility of the STR in terms of independent adjustment of mixing and aeration makes it the most
frequent configuration of choice, despite several limitations,
such as high power consumption, high shear, and problems
with sealing and stability of shafts in tall bioreactors.
Although membrane reactors (Fig. 1Cc) and packed bed
reactors (Fig. 1Ca) are advantageous in that a large amount
of cells can be immobilized per unit volume, diffusional
limitations of mass transfer to the immobilized cells, as
well as the difficulties in supplying and removing gaseous
components can limit the use of both configurations to biotransformations, where cell viability is not required.
Airlift bioreactors (ALR) using low density beads with
immobilized cells or enzymes are currently under research
for a variety of applications in bioprocess engineering,
and they have several advantages over alternate bioreactor
designs. Airlift bioreactors combine high loading of solid
particles and good mass transfer which are inherent for
three-phase fluidized beds (Fig. 1Cb). Efficient mixing in
the liquid phase is generated by air bubbles, using internal
(IL ALR) or external (EL ALR) recirculation loops (Fig.

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similar system has been developed by NASA to study the

role of physical forces on tissue formation [103]. Cells are
cultured in the annular space between two concentric cylinders, the inner one serving as a membrane for gas exchange.
Vessel rotation around the horizontal axis is used to eliminate sedimentation such that each cell or aggregate is freely
suspended at a stationary position. Rotary drum reactors
used for the cultivation of high-density plant suspensions
[104,105] showed advantages in terms of suspension homogeneity, low shear environment and reduced wall growth,
over either airlift or stirred tank reactors. Finally, plant
cells are not microbes in disguise [106], and large scale
production of embryos or shoots of elite plants in bioreactors is also important. In addition, organ cultures and mixed
root-shoot co-cultures can further improve the synthesis of
tissue-specific secondary metabolites [27,34,107,108].

11. Approaches to downstream processing

Fig. 1. Bioreactor types for plant cell, tissue and organ cultures: (A)
Mechanically agitated bioreactors: (a) stirred tank reactor equipped with
various propellers (spin, helix, bladed, paddle), (b) rotary drum tank
reactor; (B) Air driven bioreactors: (a) bubble column, (b) concentric tube
airlift reactor (IL ALR), (c) external loop airlift reactor (EL ALR), (d)
propeller loop reactor, (e) jet loop reactor; (C) Non-agitated bioreactors:
(a) packed bed, (b) fluidized bed, (c) membrane reactor.

1Bae). Relatively low shear rate, due to low relative velocity between the liquid and bubbles in cocurrent flow is
particularly desirable for shear-sensitive cell cultures. The
main advantages of airlift bioreactors are their low shear
and low energy requirements, and simple design.
Horizontal vessels, or rotary drum reactors (Fig. 1Ab)
have significantly higher surface area to volume ratio than
other reactor types [102]. As a consequence, mass transfer
is achieved with comparably less power consumption, according to Danckwerts surface renewal theory. These features are favorable for bioprocesses utilizing shear sensitive
tissues (low shear), as well as for photobioreactions (maximum exposure to light).
In bioreactors, optimal utilization of substrates and oxygen may require plug flow of gasliquid mixture without
backmixing, while efficient mixing is required to provide
uniformity of microenvironmental conditions which are essential for cell growth and well-being. Different time scales
for oxygen transfer (seconds) and cell doubling (hours to
days) can be accomodated through bioreactor design. Some
representative configurations include a horizontal rotary reactor (HRR), thin-layer tubular reactor (ThLTR) and mechanically agitated tubular reactor (MATR) (Fig. 1Ab). A

A typical recovery process involves four separation steps.

The starting feed stream contains particulate material which
must be removed, e.g. by centrifugation and/or filtration. Dilute solution of the product is then concentrated, e.g. using
non-selective separation techniques (ultrafiltration, precipitation, liquidliquid extraction, adsorption). The subsequent
steps involve a series of purifications to capture the product, and remove trace contaminants. Chromatography, in its
various forms (molecular size, charge, hydrophobicity and
molecular recognition) has proved to be the only general
separation technique that can simultaneously achieve high
purity, retain biological activity, and be scaled up to an appropriate production capacity.
A great deal of attention has been given to methods of
capturing products directly from dilute and particulate-laden
feed streams, thus eliminating the need for all concentration
steps. Affinity chromatography is such a technique, and in
cases where high specificity and sensitivity are required antibodies are an ideal choice for the separation of biomolecules
because of their high binding strength and selectivity [43].
Although extremely versatile, packed bed separations (adsorption, absorption, ion-exchange, gel and affinity chromatography) are limited to batch operation and are not capable of handling cells or particulate material. Continuous
separations can be performed using magnetically stabilized
fluidized bed (MSFB) of ferromagnetic particles or the mixture of nonmagnetic and magnetically susceptible support.
MSFB has flow and mass transfer properties of a packed
bed, in conjunction with the solid phase fluidity of a fluidized bed. Continuous separations can thus be performed
by countercurrent contact of solid and liquid phases without significant longitudinal mixing (i.e., in plug flow) at a
low operating pressure drop. Recent applications of MSFB
in biotechnology include continuous protein recovery, plant
cells filtration and plant cells cultivation [109114].

L. Sajc et al. / Biochemical Engineering Journal 4 (2000) 8999

Separations based on membranes (ultrafiltration, microfiltration, pervaporation, pertraction) have several advantages when compared to column techniques, such as
speed, easier scale up and large interfacial area available
for mass transfer. Sometimes the separation relies both on
size-discriminating ability and on the specificity of ligands
(ion-exchange or affinity) immobilized inside membrane
pores. There are three basic types of membrane modules:
flat sheet, spiral wound and hollow fiber [42].

12. Extractive bioconversion

The incorporation of an extractive step of product separation during bioconversion has been demonstrated to be
a feasible approach for improving reactor productivity by
concentrating the product in an output stream free of cells,
substrate or other feed impurities and reducing the downstream volume [67,115121]. Extractive bioconversion has
been demonstrated in laboratory scale experiments only.
Two-phase systems were used for in situ product recovery
in order to: (1) shift the equilibrium towards product formation; (2) remove products from an environment which
can cause their degradation, and (3) remove inhibitory products, i.e. increase biosynthetic rate. Extractive bioconversion
has rarely been used for products other than low molecular
weight alcohols and organic acids. However, Becker et al.
[122] have shown that extractive bioconversion can benefit
more complex products such as volatile essential oils from
plant cell cultures.
Extractive bioconversion can be carried out in two ways:
(1) an addition of the extraction solvent directly to the
bioreactor; or (2) recirculation of the whole reaction mixture through an external vessel where it is contacted with
an extraction solvent. Solvent addition into the bioreactor is simple, but may result in the formation of a stable
emulsion of organic solvent in aqueous cell medium due
to high shear rate required for solvent dispersion. Cho and
Shuler [123] have shown that these conditions are harmful
to the cells. Cell wall disruption due to extraction of some
cellular components, a blockage of nutrient diffusion due
to solvent coating of a cell, and nutrient depletion from
medium by extraction are coupled with enzyme inhibition
and change of membrane permeability by the presence of
solvent dissolved in medium. Emulsion problems can be
avoided by using columns where shear is lower and more
uniform.
Buitelaar et al. [124] have shown that plant cells were
not adversely affected by solvent when log P value (logarithm of the solvent partition coefficient over a standard
octanolwater two-phase system) was higher than 5. Bruce
and Daugulis [125] developed an extractant screening
program (ESP) with physical properties and log P values
of over 1500 solvents, which are stored in a database
for predicting partition coefficients and emulsion forming
tendencies.

95

Kolerup and Daugulis [126] have modeled extractive

bioconversion in which the solvent was added directly
to a continuously stirred tank and estimated the effect
of various process variables on the process productivity.
Roffler et al. [127] modeled extractive bioconversion in
an external extraction column in which solvent and broth
were contacted in countercurrent flow. The performance
of a differential contactor was estimated for zero and first
order product formation kinetics and backmixing in both
phases. These studies were extended to cocurrent extractive
bioconversion in an external loop airlift bioreactor with immobilized plant cells [128]. Various extraction techniques
were used to increase the production of pharmaceuticals
and biochemicals in plant cell culture: nonaqueous solvents
[41,67,122,129132], solid sorbents [122,133138] and
aqueous two-phase systems [139141].
The synthesis and excretion of secondary products are often coupled and associated with membrane transport of the
product [142]. An artificial accumulation site can therefore
reproduce similar transport phenomena in vitro. Our studies indicate that the productivity of plant cells can be improved by integration of biosynthesis and product recovery
in the extractive phase [143]. Additional advantages of integrated production and separation include enhanced rates
of mass transfer, decreased level of product inhibition, facilitated product recovery and reduced reaction volume for
a given amount of product.

13. Summary
Further studies are needed to develop correlations between
cell growth and function and hydrodynamic forces in the in
vitro culture environment, and to identify fluid dynamic parameters that are optimal for product release and separation.
Such correlations, once established, will improve our understanding of plant cell response to cultivation conditions, and
provide a basis for more exact approaches to bioreactor design and optimization. In this paper, we reviewed some of
the important scientific and engineering issues of bioreactor
design for plant cell cultivation.

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