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Appl Microbiol Biotechnol (2009) 83:809–823
DOI 10.1007/s00253-009-2049-x
MINI-REVIEW
Bioprocessing of plant cell cultures for mass production
of targeted compounds
Milen I. Georgiev & Jost Weber & Alexandre Maciuk
Received: 26 April 2009 / Revised: 16 May 2009 / Accepted: 17 May 2009 / Published online: 2 June 2009
# Springer-Verlag 2009
Abstract More than a century has passed since the first
attempt to cultivate plant cells in vitro. During this time,
plant cell cultures have become increasingly attractive and
cost-effective alternatives to classical approaches for the
mass production of plant-derived metabolites. Furthermore,
plant cell culture is the only economically feasible way of
producing some high-value metabolites (e.g., paclitaxel)
from rare and/or threatened plants. This review summarizes
recent advances in bioprocessing aspects of plant cell
cultures, from callus culture to product formation, with
particular emphasis on the development of suitable biore-
actor configurations (e.g., disposable reactors) for plant cell
culture-based processes; the optimization of bioreactor
culture environments as a powerful means to improve
yields; bioreactor operational modes (fed-batch, continuous,
and perfusion); and biomonitoring approaches. Recent
trends in downstream processing are also considered.
This paper is dedicated to Prof. Dr. Mladenka P. Ilieva on the occasion
of her 70th birthday.
M. I. Georgiev (*)
Department of Microbial Biosynthesis and Biotechnologies,
Laboratory in Plovdiv, Institute of Microbiology,
Bulgarian Academy of Sciences,
26 Maritza Blvd,
4002 Plovdiv, Bulgaria
e-mail: milengeorgiev@gbg.bg
J. Weber
Institute of Food Technology and Bioprocess Engineering,
Dresden University of Technology,
01069 Dresden, Germany
A. Maciuk
UMR CNRS 8076 BioCIS,
Laboratory of Natural Products Chemistry, School of Pharmacy,
University of Paris-Sud 11,
92296 Châtenay-Malabry, France
Keywords Bioreactor(s) . Flow cytometry . Operational
mode . Optimization . Plant cell culture . Process monitoring .
Secondary metabolite
Haberlandt’s pioneering trials
Totipotency—the ability of single somatic cells to survive
independently, divide, and ultimately form a complete plant—
is a crucial feature of plant cells that has enabled the
development of plant biotechnology. The first attempt to
cultivate plant cells outside a whole plant was described in
1902 by the Austrian botanist Haberlandt, who is justly
acknowledged as the founder of the science of plant cell
culture. In his famous paper entitled “Attempts of cultivation
with isolated plant cells” (translated from the original
German), he described a systematically organized attempt to
cultivate mesophyll, epidermis, and hair cells. Although he
did not succeed in inducing cell division, his prediction that it
should be possible to obtain a culture of dividing cells has
been the driving force of many later scientists’ careers.
Subsequent intensive investigations leading to improvements
of nutrient solutions and the discovery of plant growth
substances verified Haberlandt’s predictions (as thoroughly
summarized recently in an excellent paper by Vasil 2008) and
outlined the immense potential of in vitro cultured cells.
These advances led to the first patent for the cultivation of
plant tissue, granted to Routien and Nickell on the 29th of
May 1956, entitled “Cultivation of plant tissue” (Routien and
Nickell 1956). In their patent application, the cited authors,
in association with Pfizer & Co. Inc. (New York, NY, USA),
described the submerged cultivation of plant tissues of sorrel,
sweet clover, Agave toumeyana and crown galls isolated
from several plant species, and the possibility of producing
oxalic acid and coumarin from such cultures. In the 1960s,
810
further attempts to improve the nutrient solutions led to two
remarkable publications by Folke Skoog and co-workers, in
which two media that are still used were described:
the Murashige and Skoog medium (Murashige and Skoog
1962) and, subsequently, Linsmayer and Skoog medium
(Linsmayer and Skoog 1965). The following two decades
witnessed further developments, notably, two key events in
the field of plant biotechnology: in 1974, the Ti-plasmid of
Agrobacterium tumefaciens was discovered at Ghent Uni-
versity (Chilton 2001), providing immense opportunities for
transferring foreign genes into plant cells; and about 10 years
later, the first commercial plant cell culture-based production
system was introduced (shikonin production in a 750-L
bioreactor by Lithospermum erythrorhizon cells) by Mitsui
Petrochemical Industry.
Now, a century after Haberlandt’s pioneering research,
plant cell culture has entered the post-genomic era (the
Arabidopsis thaliana, rice, maize, and Populus trichocarpa
genomes have been sequenced to date and others are being
sequenced). During the last 5 years, 20 patents related to
plant cell cultures have been issued, and the number of
papers in the SCOPUS database with the keyword “plant
cell culture” has risen to more than 2,500. Substances
commercially produced from such cultures include
berberine, paclitaxel, ginseng saponins, and plant polysac-
charides (by Coptis japonica, Taxus spp., Panax ginseng,
Fig. 1 State-of-the-art technological platform for plant cell culture
Appl Microbiol Biotechnol (2009) 83:809–823
and Polianthes tuberosa cultures, respectively; Kieran
2001). In addition to producing plant-derived metabolites,
plant cell cultures are also being used for the large-scale
synthesis of recombinant proteins (Huang and McDonald
2009).
From field to shake-flasks and metabolite production
From prehistoric times, people have used plants for food (as
sources of proteins, carbohydrates, fats, vitamins, and
minerals) and as medicines for treating a variety of illnesses
(Kolewe et al. 2008). Over 60% of the anticancer drugs and
75% of drugs for infectious diseases currently used
originated one way or another from natural sources
(Newman et al. 2003; Cragg and Newman 2009). Further-
more, continuously increasing demands for therapeutic
molecules, produced by ever greener processes, along with
dramatic reductions in biodiversity, are driving efforts to
find alternative ways to produce high-value plant-derived
metabolites (Georgiev et al. 2007b).
State-of-the-art techniques for producing metabolites
from plant cell cultures are illustrated in Fig. 1. Today, it
is possible to induce callus cultures from virtually any plant
species, although some plants (especially monocotyledons)
are more recalcitrant than others. The German Collection of
Appl Microbiol Biotechnol (2009) 83:809–823
Table 1 Recent reports on applications of various elicitors for increasing metabolite yields
Elicitora Plant cell culture
Vanadyl sulfate (25 mg/L) Lavandula vera
Methyl jasmonate (50 μM)
2-hydroxyethyl jasmonate (200 μM) Panax notoginseng
TFINA (100 μM) Taxus chinensis
DPCEJ (100 μM)
S-methyl benzo-1,2,3-thiadiazole-7-
carboxylates (μL/mL)
Chitosan (50 mg/L) Azadirachta indica
Aspergillus niger filtrate (100 mg/L) Mentha piperita
Additional examples can be found in a recent review by Smetanska 2008
TFINA trifluoroethyl 2,6-dichloroisonicotinate, DPCEJ 2-(2,6-dichloro-pyridine-4-carbonyloxy)-ethyl jasmonate
a
Elicitor concentrations are given in parenthesis
Microorganisms and Cell Cultures, for example, maintains
over 700 plant cell lines from 80 plant families (www.dsmz.
de). The most important parameters from an economic
perspective are the yield of desired metabolite(s) and/or the
volumetric productivity of the plant in vitro system.
Generally, secondary metabolites account for less than 1%
of the dry weight of plant cells, and sometimes much less
(e.g., the paclitaxel content in Taxus bark is only 0.01% of
dry weight), thus improving yields of desired metabolites is
a key task for which several strategies have been developed
and successfully applied (Fig. 1, right side). Firstly, due to
the heterogeneity of callus cultures, the selection of highly
productive cell lines is a traditional (and essential) step
towards cost-effective production, and various treatments
have been used to select lines with desired attributes. For
example, Georgiev et al. (2006b) used two analogs of
phenylalanine (m-F-D,L-phenylalanine and p-F-D,L-
phenylalanine) as agents to select Lavandula vera cell lines
with increased levels of phenylalanine ammonia lyase
(PAL, a key enzyme in phenolics biosynthesis). These
analogs killed most wild type cells, but not those that
strongly expressed PAL. Resistant cells were then isolated,
and when cultivated in liquid medium, they yielded
approximately 1.5-fold higher volumetric yields of
rosmarinic acid than cultures of unselected cells. The
selected cell lines stably maintained their enhanced biosyn-
thetic potential, and even after ten passages, their rosmarinic
acid content remained unchanged (Georgiev et al. 2006b).
However, it should be noted that in several cases, selected
cell lines have tended to revert to unselected-like states and
lose their enhanced biosynthetic potential (Verpoorte et al.
2002).
Several strategies have also been applied to boost the
yield of targeted metabolites at the cell suspension stage.
811
Targeted Fold Reference
metabolite increase
Rosmarinic acid 2.8 Georgiev et al. 2006a, 2007a
2.4
Ginsenoside (total) 5.7 Wang et al. 2006
Taxuyunnanine C 1.6 Qian et al. 2006
2.6 Qian et al. 2006
3.4 Xu et al. 2006
Azadirachtin 2.8 Prakash and Srivastava 2008
Menthol 1.8 Chakraborty and
Chattopadhyay 2008
Optimization of the nutrient medium is a frequently used
approach here, as it is in microbial biotechnology. Usually
the medium composition is modified with respect to the
concentrations and/or ratios of main nutrients (carbon,
nitrogen, and phosphorous sources). For instance, using a
modified simplex method, Pavlov et al. (2000) optimized
the nutrient medium for cultivating L. vera cell suspen-
sions, leading to a 27-fold increase in rosmarinic acid
production. By applying another statistical approach,
response surface methodology, Hanchinal et al. (2008)
optimized the medium components for suspension cultures
of Daucus carota in terms of both growth and the
production of the targeted metabolite β-carotene (1.4-fold
compared to the standard medium). Various elicitation
strategies, which fundamentally exploit natural processes,
namely, the specific plant’s response cascades to stress
factors, provide further potentially powerful ways to
improve yields and productivities of plant cell cultures.
Elicitors include chemicals (inter alia heavy metal ions and
various compounds) and biofactors (including microorgan-
isms, herbivores, and plant cell wall components) that can
induce physiological changes in the target living organism
(Zhao et al. 2005; Matkowski 2008). Table 1 summarizes
some recent reports on improvements of yields achieved
through elicitation strategies. Use of vanadyl sulfate, which
boosts rosmarinic acid contents in lavender cells, will
increase process costs by approximately 25 € (at the most
efficient concentration of 25 mg/L; Georgiev et al. 2006a)
for thousand-liter scale production. Other reports draw
attention to novel chemically synthesized elicitors, e.g., 2-
hydroxyethyl jasmonate (Wang et al. 2006), isonicotinic
acid derivatives (2,6-dichloroINA, a synthetic functional
analog of salicylic acid; Qian et al. 2006), and S-methyl
benzo-1,2,3-thiadiazole-7-carboxylate derivatives (Xu et al.
812
2006), which have effectively enhanced yields of secondary
metabolites. Although the prices of these synthetic com-
pounds are roughly approximately five times higher than
the starting, commercially available methyl jasmonate and
2,6-dichloroisonicotinic acid (Drs. Zhong, Xu, and Qian,
personal communications), their addition would not signif-
icantly increase the overall production costs.
Metabolic engineering approaches are also applied to
identify mechanisms regulating the production of targeted
metabolites in attempts to boost yields. However, since
many pathways involved in secondary metabolite biosyn-
thesis have not yet been fully defined, elucidating the genes
involved and their control elements is a serious challenge
(Kolewe et al. 2008). Delivering genes encoding key
enzymes for targeted secondary metabolite production by
the T-DNA of A. tumefaciens (the causative agent of crown
gall disease), plant viral vectors, particle bombardment, or
electroporation of protoplasts is another powerful strategy
to increase yields (Vasil 2008). Kiselev et al. (2007), for
example, observed a 100-fold increase in resveratrol
production in calli of Vitis amurensis transformed by the
rolB gene of A. tumefaciens (under the control of the
cauliflower mosaic virus 35S promoter).
Synergistic application of the so-called enhancement
strategies mentioned above can result in approximately
1,000-fold improvements in yields of targeted metabolites
(Smetanska 2008) and deliver volumetric yields of up to
several grams per liter of culture (production levels that
validate the “plant cell factory” concept). Besides genes
designed to improve metabolite yields, genes for producing
recombinant proteins are frequently delivered to host plant
cells. However, since such applications are beyond the
scope of this review and were recently summarized (Huang
and McDonald 2009), they will not be further considered
here.
The left side of the scheme in Fig. 1 presents a relatively
novel stream of strategies for boosting yields and produc-
tivity, the so-called “omics” approaches. Recent advances
in techniques and instruments (e.g., in complementary
DNA (cDNA)-amplified fragment-length polymorphism,
real-time-polymerase chain reaction, two-dimensional gel
electrophoresis, gas chromatography-mass spectroscopy,
liquid chromatography-mass spectrometry (LC-MS), liquid
chromatography-solid phase extraction-nuclear magnetic
resonance, and capillary electrophoresis analysis methods
and instruments) have enabled comprehensive investigation
of plant systems (Oksman-Caldentey and Inze 2004). The
functional genomic approach (including transcriptomics
and proteomics), synergistically coupled with metabolo-
mics, forms a system biology approach, which may allow
the biochemical machinery of plants to be fully explored
(Goossens and Rischer 2007) and (hence) their biosynthetic
potential to be fully exploited. For instance, recent attempts
Appl Microbiol Biotechnol (2009) 83:809–823
to elicit Catharanthus roseus cells with methyl jasmonate
enabled the isolation of 417 gene sequence tags (using
cDNA-amplified fragment length polymorphism analysis)
and 178 metabolites (by LC-MS) and further establishment
of gene-to-gene and gene-to-metabolite networks related to
the biosynthesis of terpenoid indole alkaloids (Rischer et al.
2006; Goossens and Rischer 2007). In addition, a metab-
olomics approach alone has revealed significant differences
in metabolic profiles between shake-flask and stirred tank
reactor Rosa damascena cell suspensions (Pavlov et al.
2005c). Connection of “omic” analyses with assays for
evaluating biological activities (e.g., anticancer or anti-
inflammatory activities) would facilitate the discovery of
new drug candidates with improved biological activities. In
this context, it is worth mentioning that only half of the ca.
100,000 plant secondary metabolites have been structurally
elucidated to date (De Luca and St Pierre 2000; Oksman-
Caldentey and Inze 2004).
Vessels hosting the plant cells—hardware configuration
An essential step towards the biotechnological production
of desired compound(s) is the transfer of biosynthetic
processes from shake-flasks to bioreactors. Such transfer,
while maintaining the biosynthetic potential of the plant
cell cultures, is not always straightforward. The main
difficulties arise from the dependence of the biosynthesis
of target compounds on several inter-related factors, of
varying significance, depending on the method of cultiva-
tion and physiological peculiarities of the source plant.
A wide variety of bioreactor designs have been used for
cultivating plant cell cultures. The traditional bioreactor
configurations (e.g., stirred tank reactors, airlift reactors,
and bubble column reactors) used for in vitro cultivation of
plant cells have been copied from those developed and used
in microbial biotechnology. The first dedicated attempts for
cultivating plant cell cultures in bioreactors, at the end of
the 1970s, heightened awareness of the high shear
sensitivity of plant cells in vitro, and, consequently, only
air lift reactors were considered suitable for cultivating
them for more than a decade (Verpoorte et al. 2002).
However, numerous laboratory studies performed with
stirred tank reactors, in combination with the first commer-
cial systems, subsequently challenged such perceptions.
Indeed, the world’s largest plant cell culture facility, created
by Diversa (situated in Ahrensburg, Germany), has a
cascade of five stirred tank bioreactors (75, 750, 7,500,
15,000, and 75,000 L). Ritterhaus et al. (1990) reported the
use of the cascade for large-scale cultivation of Echinacea
purpurea, Rauwolfia serpentina, and other plant cell
cultures. Today, this current good manufacturing practices
(cGMP) facility (i.e., one that meets current United States
Appl Microbiol Biotechnol (2009) 83:809–823
Department of Agriculture good manufacturing practice
regulations) is used to produce paclitaxel (the active
ingredient of Bristol–Myers Squibb’s Taxol® drug for the
treatment of breast, ovarian, and other forms of cancer) by
Phyton Biotech Inc. (owned by DFB Pharmaceuticals;
www.phytonbiotech.com). Airlift and bubble column
reactors have also been promoted for cultivating photoau-
totrophic or photomixotrophic cell suspensions and stress-
sensitive cultures due to the low shear environment they
provide.
Wang and Zhong (1996) developed a new type of
bioreactor for shear-sensitive plant in vitro systems named
the centrifugal impeller bioreactor (CIB). Agitation in this
system is based on the same principles as a centrifugal
pump, involving use of a centrifugal-pump-like impeller in
a conventional vessel. Recently, Zhang and Zhong (2004)
used a 3-L CIB for cultivating Panax notoginseng cell
suspensions and found that at an agitation speed of
145 rpm, the accumulated biomass reached 22 g/L. Further,
the process was successfully up-scaled to a 30-L CIB
(working volume, 21 L), in which the accumulated biomass
exceeded 25 g/L, and the volumetric yields of saponins and
polysaccharides were also higher (1.7 vs 1.5 g/L and 2.9 vs
2.7 g/L, respectively). Based on these results, Zhang and
Zhong (2004) concluded that CIB might have great
potential for efficient mass production of desired com-
pounds from large-scale, high-density plant cell cultures.
Successful scale-up of Azadirachta indica suspension
culture for azadirachtin production was done in a stirred
tank bioreactor equipped with centrifugal impeller and
compared with similar bioreactor with a setric impeller. The
CIB demonstrated less shearing and improved O2 transfer
than the stirred tank bioreactor equipped with setric
impeller with respect to biomass and azadirachtin produc-
tion (Prakash and Srivastava 2007).
In order to minimize validation efforts and production
costs under cGMP regulations, several disposable bioreac-
tor designs for plant cell cultures have been developed
recently (e.g., life reactor, ebb-and flow-bioreactor, plastic-
lined bioreactor, wave reactors, Nestlé’s wave and under-
tow bioreactor, and slug bubble bioreactor). Wave reactors
Fig. 2 Disposable bioreactors
for cultivating plant cell
cultures. a Wave bioreactor
(redrawn from Eibl and Eibl
2008). b Slug bubble reactor.
c Wave and undertow reactor
(redrawn from Terrier et al.
2007)
813
(Fig. 2a) are mechanically driven reactors in which there is
no direct mechanical agitation (Eibl and Eibl 2002), making
them suitable for cultivating highly shear-stress sensitive
plant cell lines. Instead, they provide a wave motion,
generated via rocking (with rocking rates and angles that
can be varied according to the sensitivity of the cultured
line), thereby ensuring agitation and bubble-free aeration.
They also have a plastic disposable chamber, which
facilitates the fulfillment of cGMP regulations (Eibl and
Eibl 2006). Recently, Eibl et al. (2009) reportedly achieved
biomass productivity of 40 g fresh biomass/L/day with a
doubling time of approximately 48 h during the cultivation
of Vitis cells in BioWave® reactors. In addition, high levels
of paclitaxel productivity have been observed in Taxus
baccata cells, cultured in BioWave® reactors, amongst the
highest reported to date (Bentebibel et al. 2005; Eibl et al.
2009). In 2006, the same manufacturer (Wave Biotech
AG®, Tagelswagen, Switzerland) released a production-
scale commercial model with a maximum working volume
of 300 L named the BioWave® 600, in which the
temperature, rocking rate, and rocking angle can be varied
in ranges of 15–45°C, 2–16 rocks/min and 4–10°, respec-
tively. Furthermore, the absence of air bubbles and wall-
growth, as well as the reduced foaming seems to make
these devices quite suitable for cultivating plant cell, tissue,
and organ cultures (Eibl et al. 2009).
A group from Nestle R&D Centre in Tours, France,
recently reported another two new types of disposable
bioreactors (the wave and undertow bioreactor and slug
bubble bioreactor; Fig. 2b, c). The wave and undertow
reactors, with working volumes ranging from 10 to 100 L,
are made of biopharmaceutical grade flexible polyvinyl
chloride (allowing autoclavation) and provide a wave/
undertow motion that ensures mixing and bubble-free
aeration of the culture (Terrier et al. 2007). The system is
placed on a horizontal surface, equipped on one side with a
moveable platform, which rises and falls, creating waves
followed by an undertow. Although the volumetric mass
transfer coefficient was lower than in a conventional 10-L
stirred tank reactor, in tests with Glycine max cell cultures,
the accumulated mass in a 30-L wave and undertow reactor
814
was approximately 1.3-fold higher than in a stirred tank
reactor, and doubling times of 2.2–3.2 days were achieved
(Terrier et al. 2007). The other type of reactor described by
this group, the slug bubble reactor, provides working
volumes from 10 to 70 L. These reactors consist of a
vertical, flexible plastic cylinder (made of biopharmaceut-
ical grade polyethylene), in which aeration is achieved via
the generation of large cylindrical bubbles, which move
from the bottom to the top of the reactor. Here again, the
volumetric mass transfer coefficient was found to be lower
than in a compared stirred tank reactor, but during the
cultivation of a Nicotiana tabacum cell suspension culture,
the accumulated biomass was approximately 1.2-fold
higher than in the stirred tank reactor. In addition, the
isoflavone content of G. max cells cultivated in both
bioreactors was higher than in the stirred tank reactor
(Terrier et al. 2007). Recently, this group reported further
increases of reactor volumes to 750 L for wave and
undertow and to 175 L for the slug bubble reactor (Ducos
et al. 2009). These recently introduced types of bioreactors
(especially wave, and wave and undertow reactors) may be
suitable for the cultivation of highly shear-stress sensitive
cell lines.
Besides the need for large-scale culture for production
purpose, the development at an early stage of small scale
cultivations systems allowing high throughput is of consid-
erable interest for tasks like cultures screening, media
development, investigations of the effects of basic process
variables (e.g., the inoculum ratio and temperature), and the
acquisition of fundamental kinetic data (Büchs 2001).
Although small scale cultivation is not a new technology
(e.g., shake-flasks have been used for cultivation for
decades), their application was hampered by the partially
undefined cultivation condition (e.g., oxygen limitation)
and the lack of on-line measurements that are mandatory
for high throughput. In recent years, a number of
cultivations systems (with working volumes ranging from
100 μL to several milliliter), allowing small scale cultures
under defined conditions and defined engineering parame-
ters, has been developed (Anderlei et al. 2004; Samorski et
al. 2005). Among others, shaking flasks and shaken
microtiter plates (MTP) are used as vessels. For shaking
flasks, the “respiration activity monitoring system”
(RAMOS) measuring the oxygen transfer rate (OTR) and
the carbon dioxide transfer rate on-line was developed
(Anderlei et al. 2004; HiTec Zang GmbH, Herzogenrath,
Germany and Kühner, Birsfelden, Switzerland).
The BioLector represents an MTP cultivation system
(commercialized by m2p-labs, Aachen, Germany) that
copes with the demands defined above. The light scattering
signal captured by this device was found to correlate very
well to the biomass concentration in microbial fermenta-
tions (Samorski et al. 2005). Besides, this cultivation
Appl Microbiol Biotechnol (2009) 83:809–823
system is capable to monitor several fluorescences (e.g.,
reporter proteins and nicotinamide adenine dinucleotide)
on-line. Here, the fact that the shaking is not interrupted
during the measurements not only ensures continuous mass
transfer, but also homogenizes the suspended cells. There-
fore, the light scatter and fluorescence measurements are
suitable for heterogeneous and large cells aggregates of
plant suspension cultures. Figure 3 shows an exemplary
cultivation of N. tabacum CV BY2 HAS in the BioLector,
evaluating the influence of different fractions of inoculum
on the biomass accumulation at a 500-µL scale. In addition,
orbitally shaken non-instrumented tubes (e.g., TubeSpin
bioreactor, CultiFlask 50 disposable bioreactor) have been
successfully used for plant cell suspension-based process
developments.
Bioreactor internal environment optimization
and operation modes
Plant cells are much bigger than microorganisms, with sizes
of 40–200 μm, and they have a rigid cellulose-based cell
wall. They also have one or more large, distinct vacuoles,
which may occupy 90–95% of their volume. Nutrients,
metabolites, waste products, and water are generally stored
in the vacuole, forming a fluid that is far less viscous than
the cytoplasm. These large “bags of water with thin cell
walls” (Verpoorte et al. 2002) are much more sensitive to
shear forces than the smaller microbial cells. Plant cells also
grow more slowly than microorganisms, with doubling
times of 2–5 days. Normally, the broth of plant cells
exhibits Newtonian behavior, but often during their
cultivation, significant amounts of extracellular polysac-
charides (EPS) are observed, forming highly viscous, non-
Fig. 3 Cultivation of Nicotiana tabacum CV BY2 HAS in the
microtiter plate cultivation system BioLector (48 well plate, 26°C,
800 rpm, 3 mm shaking diameter, and 500 µl volume) with different
fractions of inoculum: 20% (closed circles), 40% (open circles), and
60% (closed triangles)
Appl Microbiol Biotechnol (2009) 83:809–823
Newtonian broths. For instance, Haas et al. (2008) reported
very high EPS production during the cultivation of
Helianthus annuus cells, reaching levels of 1.9 g/L at day
9 in a 5-L stirred tank reactor. Another important feature of
plant cells is their tendency to form aggregates, consisting
of several hundred cells, in clumps of 0.5 cm diameter or
more (H. annuus cell suspension cultures tend to form huge
aggregates with diameters of 1.0–1.2 cm; Georgiev,
unpublished data). The aggregates also tend to settle
readily; for example, in the mentioned H. annuus bioreactor
cultivation, the cells/aggregates fully settled approximately
20 min after ending agitation and aeration. Wall-associated
growth and flotation of the cells also frequently occur
during the bioreactor cultivation of plant cells and may
hamper biosynthetic processes. These features of plant cells
have detrimental effects on the mass transfer of potentially
limiting substrates (oxygen and the carbon source) to the
cells and/or secretion of potentially inhibiting (by-)products
from them.
Agitation
Agitation is an essential process during the bioreactor
cultivation stage, since it promotes homogeneity with
respect to the plant cells’ mass and nutrients, and enhances
mass and heat transfer in bioreactors. Generally, for plant
cell cultivations, bioreactors can be divided into three types
according to their mode of agitation: mechanically driven,
pneumatically driven, or combined. Mechanical agitation is
the most intensive and, thus, the most frequently applied.
Since plant cell cultures are very sensitive to mechanical
stress (usually referred as shear), several impeller designs
have been developed besides the well-known Rushton
turbine and propeller impellers—including paddles,
anchors, helical ribbon impellers, and spin stirrers, as
comprehensively reviewed by Doran (1999)—in attempts
to meet the specific physiological requirements of plant
cells. However, each change in bioreactor design that
increases its complexity raises overall process costs. The
tip speed (Ts) is a parameter that is used in the scale-up
process and is frequently used as a proxy measure of shear
stress.
Ts ¼ p
N
Di ðm=sÞ; ð1Þ
where N is the rotational speed of the stirrer (1/s) and Di is
the impeller diameter (m). Pavlov et al. (2005a) found the
optimal tip speed for growth of L. vera MM cell suspension
culture in a 3-L propeller stirred tank reactor to be 0.63 m/s,
while the highest volumetric yields of rosmarinic acid were
obtained with a tip speed of 0.95 m/s. For a suspension
culture of Perilla frutescens in bioreactor with marine
impeller, the maximum cell concentration, the anthocyanin
815
pigment production, and productivity were all relatively
higher at an average shear rate of 20–30 (1/s) or an impeller
tip speed of 0.5–0.8 m/s (Zhong et al. 1994a). Recently,
Haas et al. (2008) reported for a cell suspension culture of
H. annuus, grown in a 5-L stirred tank reactor (equipped
with a two six-blade Rushton turbine), that the highest
biomass was accumulated at a tip speed of 4.08 m/s. In
addition, Chisti (1999a) found that impeller tip speeds
exceeding approximately 2.36 m/s cause damage to cell
suspension cultures of C. roseus and N. tabacum. These
studies confirmed that the susceptibility to shearing forces
varies widely amongst cell cultures. Moreover, even for the
same culture line, the age of the culture and the cultivation
regime may significantly influence cells’ responses to shear
fields (Chisti 1999a).
Aeration
Aeration of plant cell cultures fulfills three main functions:
maintenance of aerobic conditions, desorption of volatile
products, and removal of metabolic heat. The specific O2
uptake rate (respiration) of plant cells depends on the cell
culture line, cultivation conditions, and growth phase, but is
generally of the order of 6.10−4 g O2/g dry cell weight/min
(Chisti 1999a; Kieran 2001). The OTR must be sufficiently
high to provide enough oxygen to comply with the
respiratory demands of the cells, and, therefore, supporting
the growth of the cells and production of desired com-
pounds, but not too high, since both excessive and
insufficient oxygen supply can hinder cell growth and
secondary metabolism. In order to avoid oxygen limitation,
the dissolved oxygen has to be kept above the critical
oxygen concentration that was reported to be 15–20% of air
saturation (Kieran 2001). Pavlov et al. (2005a) studied the
influence of dissolved oxygen concentration across the
range 10–50% of air saturation (dO2, varied by adjusting
air/nitrogen ratios while keeping the agitation speed
constant) on the growth of L. vera cells and their
production of rosmarinic acid, and established that
the highest biomass amounts and volumetric yields of
rosmarinic acid were attained at 50% and 30% of air
saturation, respectively. Significant reductions in both
growth and production of secondary metabolism were
observed at 10% dO2, which supports the observations
made by Kieran (2001). Active control of dO2 within target
limits (by aeration at fixed agitation speed) might be
advantageous for extremely shear-sensitive cell lines.
However, dO2 is not a parameter that could be used in
up-scaling a process; such parameters include (inter alia)
the volumetric mass transfer coefficient (KLaL) and the air
flow rate (liter/liter/minute).
dCL =dt ¼ KL aL ðC   CL Þ; ð2Þ
816
where C* is the saturation concentration, CL is the
dissolved oxygen concentration at time t, KL is the mass
transfer coefficient, and aL is the gas-liquid interfacial area
per unit volume of the liquid (Chisti 1999b). Several
methods are available for measuring KLaL values, the most
common being dynamic gassing-in, oxygen balance, and
sulfite-oxidation methods (Chisti 1999b). The mass transfer
coefficient is a function of both aeration and agitation, thus,
initial KLa values have been measured for various cultiva-
tion systems. Zhang and Zhong (2004) found that the initial
KLa is a key factor affecting cell growth and production of
ginseng saponins and polysaccharides in the high-density
cultivation of P. notoginseng cells in a 3-L CIB, in which
the highest productivity was achieved at 30.2/h. Neither
higher (86.4/h) nor lower (15.7/h) initial KLa values were
beneficial to the production of ginseng saponins and
polysaccharides by the bioreactor cell cultures. Further,
KLa was used for up-scaling the process from a 3-L CIB to
a 30-L CIB (Zhang and Zhong 2004). The ideal air flow
rate for plant cell suspension cultures is generally assumed
to be in the range of 0.1–0.5 L/L/min (Eibl and Eibl 2008);
however, incremental increases in the flow rate during their
cultivation to match the physiological state of the cells
could be advantageous. By applying such an aeration
scheme (with a flow rate of 0.5 L/min until day 2 of
cultivation and 1.0 L/min thereafter), Haas et al. (2008)
established high growth of H. annuus cells, allowing the
accumulation of up to 15 g dry biomass/L.
Temperature
According to van’t Hoff’s law, an increase in temperature
will cause an increase in the rate of endothermic reactions.
Hence, an increase in the temperature should enhance the
speed of conversions in the plant cells and contribute to the
economy of the process. However, since enzymes (which
catalyze the reactions) are proteins, excessive increases in
temperatures will lead to their denaturation, and hence,
reduce the speed of the reactions they catalyze. Therefore,
there is an optimum temperature for any biological
conversion process. The cultivation temperature influences,
to some extent, both the growth of the plant cell culture and
the biosynthesis of the desired metabolites. Published
results (Zhao et al. 2001; Ten Hoopen et al. 2002; Georgiev
et al. 2004) have shown that the optimal temperature
(generally 23–30°C) depends on the plant species, and even
for the same species, the optimal temperatures may vary.
This is mainly because the metabolites’ biosynthesis is
regulated by key enzymes, which have different tempera-
ture optima. Ten Hoopen et al. (2002) found that the
optimal temperature for growth of C. roseus suspension
cultures and the production of ajmalicine from them was
27.5°C, at which, both variables were approximately 2.3-
Appl Microbiol Biotechnol (2009) 83:809–823
fold higher than at 25°C. The cited authors also found that
tryptophan decarboxylase (a possible key enzyme in the
ajmalicine pathway) activity was maximal at this temper-
ature. However, for cell suspension cultures of L. vera, it
was found that the optimal temperature for growth was 30°C
(33.2 vs 31.8 g/L at 26°C), while the volumetric production
of rosmarinic acid was highest at 28°C (approximately 1.7-
fold than at 26°C; Georgiev et al. 2004). This is an important
observation with respect to optimizing the temperature
regime for a production process, indicating that a two-stage
process may be beneficial, with an initial stage during which
conditions are optimized for growth, and a second stage in
which they are adjusted to maximize the production of
desired compound(s). However, it should be stressed that
fine-tuning temperature so closely in full-scale production
would be difficult since temperature gradients frequently
appear at higher bioreactor scales.
Optimization of culture conditions
Optimizing the internal environment of the bioreactor is
essential in order to maximize the cost-effectiveness and
economic feasibility of any bioprocess. Nevertheless, in
contrast to microbial biotechnology, where optimization is a
routine procedure (Ha et al. 2007), optimization of culture
conditions for plant cell cultures has been poorly investi-
gated. However, Pavlov et al. (2005b) recently developed
and applied polynomial regression models for describing
rosmarinic acid production in a 3-L stirred tank reactor as a
function of the dO2 concentration, agitation, and tempera-
ture, followed by statistical optimization using a modified
simplex method. The outcome was a significant enhance-
ment of the volumetric yields of rosmarinic acid (to
approximately 3.5 g/L, twofold higher than in the shake-
flask stage). The optimal culture conditions identified (50%
of air saturation, 400 rpm, and 29.9°C) are unusual for
cultivation of plant cell suspensions, and the enhanced
rosmarinic acid content in the lavender cells (approximately
13% of cell weight) is most likely a response to the elevated
stress levels. However, this attempt demonstrates that
optimization of the basic operational parameters is a
powerful strategy to enhance biomass accumulation and/or
targeted metabolites’ yields.
Fed-batch, continuous, and perfusion processes
A major disadvantage of batch processes is that significant
amounts of time are taken up by system and media
sterilization, filling and emptying, and cleaning the system.
Thus, to improve the cost-effectiveness of culturing plant
cells, various operational modes have been developed,
including fed-batch, repeated fed-batch, semi-continuous,
and continuous cultivation.
Appl Microbiol Biotechnol (2009) 83:809–823
Fed-batch operation involves the addition of one or
more nutrients continuously or intermittently to the initial
medium after the start of cultivation, or from some point
during the batch process. Basically, fed-batch operation can
be sub-divided into two basic modes: with and without
feedback control (Harada et al. 1997). Fed-batch operation
without feedback control was applied by Luo et al. (2002)
in a study of the effects of different carbon sources in the
feeding medium (sucrose, glucose, fructose, and maltose)
and varying the feeding time (feeding on days 8, 12, 16,
and 20 of cultivation) on the growth of Taxus chinensis
cells and production of paclitaxel in a 5-L stirred tank
reactor. The highest amounts of biomass (approximately
1.5-fold higher than in a batch process) were accumulated
when sucrose-containing medium was added on day 8 of
cultivation, while the highest volumetric paclitaxel yields
(approximately 4.9-fold higher than in the batch process)
were achieved when sucrose-containing medium was added
on day 16 of cultivation. The productivity of paclitaxel
(milligram/liter/day), overall throughout the process, was
3.8-fold higher in fed-batch than in batch cultivation mode.
Hu et al. (2001) developed another fed-batch operational
mode in which sucrose was fed just prior to a sharp
decrease in the specific oxygen uptake rate (>0.20 mmol/g
DW/h). By applying this feeding policy to P. notoginseng
cell suspensions, cultivated in a 10-L airlift bioreactor (Hu
et al. 2001) and a 30-L CIB (Zhang and Zhong 2004), very
high biomass productivity of approximately 1.5 g/L/day
was achieved, and both saponins and polysaccharide
productivities were also higher than in a comparative batch
process.
Theoretically, a continuous process can be described
with the following equations:
dx=dt ¼ ðm  DÞX ð3Þ
D ¼ F=V ; ð1=hÞ ð4Þ
TR ¼ V =F; ðhÞ ð5Þ
MS ¼ RS  m=YSX ; ð6Þ
where μ is the specific growth rate (1/h), D is the dilution
rate (1/h), X is the biomass concentration (g/L), F is the
feed flow (L/h), V is the volume of the bioreactor (L), TR is
the residence time (h), MS is the maintenance value (C-mol/
C-mol/h), RS is the rate of substrate consumption (C-mol/C-
mol/h), and YSX is the yield of biomass per unit mass of the
carbon source (C-mol/C-mol). When D = μ = constant, then
dx/dt = 0, or a stationary state is established; in transient
state, if μ > D, the biomass in the bioreactor increases,
817
while if μ < D, the biomass in the bioreactor decreases due
to cell washout. The efficacy is likely to be highest if D is
approximately equal to μmax. Current methods for contin-
uous cultivation include variants without feedback control
(e.g., in chemostats, where the substrate is fed at a constant
rate) and with feedback control (e.g., in turbidostats, where
the turbidity of the culture is kept constant by adjusting the
rate at which substrate is fed, and auxostats, where the pH
or dO2 of the medium is maintained at a set value).
However, regardless of the method chosen for continuous
cultivation, it should be confirmed that steady-state behav-
ior has been reached (Van Gulik et al. 2001), which for
plant cell suspensions is assumed to take approximately two
to four residence times (Eq. 5). Van Gulik et al. (1992)
performed experiments on continuous chemostat cultures of
C. roseus and N. tabacum and examined the growth
stoichiometry of both species in steady-state glucose-
limited chemostats across a wide range of dilution rates
(0.0036–0.0177/h). Relatively large deviations between
both yield and maintenance data and the regression line of
substrate consumption were found, which may have been
due to variations in the cells’ biomass composition (arising
from the formation of biochemically inactive storage
compounds, such as polysaccharides). The phosphorous
level was found to be a major determinant of the formation
of storage compounds, because when there was sufficient
phosphate, the carbon source was used for biomass
production, but if phosphate was limiting, the carbon
source was converted into storage compounds (Van Gulik
et al. 1992, 2001). Based on this observation, Van Gulik et
al. (1993) developed a structural model, which divides the
biomass into active biomass and storage products (consid-
ered as inactive biomass). The validity of the model was
further supported by good agreement between model
predictions and experimental results under entirely different
culture conditions in steady-state chemostat culture (Van
Gulik et al. 1993). Van Gulik et al. (2001) reviewed
continuous cultivation attempts with different plant cell
cultures and noted that reported critical dilution rates vary
between 0.0022/h for cell suspensions of Phaseolus
vulgaris to 0.0277/h for cell suspensions of C. roseus, and
biomass yields per unit mass carbon source vary between
0.36 (C-mol/C-mol) for cell suspensions of Acer pseudo-
platanus to 1.58 (C-mol/C-mol) for cell suspensions of C.
roseus. The cited authors concluded that the yields achieved
for plant cells are in the same range, or even slightly higher,
than those obtained using microorganisms. However, the
maintenance values (Eq. 6) were evidently lower for plant
cells.
Perfusion cultivation is carried out by continuously
feeding fresh medium to the bioreactor and constantly
removing the cell-free spent medium while retaining the
biomass in the reactor. The key parameter for successful
818 Appl Microbiol Biotechnol (2009) 83:809–823

perfusion operation is the cell retention efficiency (Su and

Arias 2003). Perfusion allows to achieve higher biomass
concentration than in continuous cultivations without cell
retention, separation of the toxic (by-)products from the
active cells, and is advantageous when the targeted
metabolite is extracellular. Su and Arias (2003) grew
Anchusa officinalis cells in a perfusion bioreactor with
perfusion rates up to 0.4 L/L/day, with complete cell
retention, and found the dry cell weight exceeded 20 g/L,
and the packed cell volume exceeded 70%. De Dobbeleer
et al. (2006) coupled a column filled with XAD-7 to the
outlet of a perfusion reactor for cultivating Eschscholtzia
californica achieving a doubling time of approximately
67 h. Although further developments with respect to
optimization of the culture conditions are required,
coupling perfusion bioreactor systems with appropriate
adsorption columns might have great potential for the
continuous extraction of targeted compounds. Su and
Arias (2003) concluded that perfusion bioreactor systems
showed high potential as an alternative to immobilized
plant bioreactors.

Monitoring culture growth

In biotechnological processes, cells serve as “factories” that

convert substrates into products. Hence, their concentration
has a major impact on many aspects of the processes, and
monitoring cell growth during the cultivation is essential
(especially during large-scale processes). Timely informa-
tion on the physiological status of plant cells allows more
effective control and management of the biosynthetic
processes. However, obtaining accurate measurements of
cell growth in bioreactors has been difficult, in large part
due to the tendency of the cell to aggregate and their
significant heterogeneity. Traditional techniques include
gravimetric determination of fresh and dry cell weight,
and measurements of packed and settled cell volumes, and/
or cell numbers. Significant disadvantages of these methods
are their time-consuming nature and the impossibility of
Fig. 4 a Relationship between changes in medium conductivity and
using them for on-line monitoring. A more prompt measure
biomass concentration during the cultivation of Harpagophytum
can be obtained by exploiting the known linear relationship procumbens cell suspension in shake-flasks (open squares) and
between changes in medium conductivity and cell growth. Lavandula vera in a 3-L stirred tank reactor (closed circles; redrawn
To illustrate the utility and accuracy of this analysis, linear from Pavlov et al. 2005a). b Proportions of the cell population in the
G0 +G1 (open square), S (open circle) and G2 +M (closed square)
correlations observed between increases in cell mass and
phases during batch cultivation of Helianthus annuus cell suspension
reductions in medium conductivity for cell suspension culture in a 5-L stirred tank reactor (reprinted from Haas et al. 2008
cultures of Harpagophytum procumbens cultivated in with permission of Zeitschrift fuer Naturforschung). c Linear
shake-flasks and L. vera cultivated in 3-L stirred tank relationship between dry cell weight and proportions of the cells in
the G0 +G1 phase. d Histogram of relative DNA contents of the reactor
reactor are presented in Fig. 4a. The high calculated
culture at the sixth day of cultivation
correlation coefficients (r2 0.95–0.99) and those reported
by Haas et al. (2008) for H. annuus cell suspensions in a
5-L stirred tank reactor (r2 0.99) prove the reproducibility
and reliability of this method for fast (compared to the
Appl Microbiol Biotechnol (2009) 83:809–823
widely used methods), effective, and noninvasive moni-
toring. James and Lee (2000) reported another method for
fast monitoring of N. tabacum cells’ growth based on
optical density measurements. By combining measure-
ments from horizontal and vertical reading frames, the
authors prevented large errors and unstable readings due
to the cells settling. The calibration curve between
absorbance at 632 nm and dry cell mass showed a very
high correlation coefficient (0.99), with total procedure
durations of about 5 min, allowing indirect real-time
determination of cell mass. The cell concentration of P.
frutescens was successfully measured on-line and in situ
with a laser turbidimeter (at 780 nm; Zhong et al. 1993).
Furthermore, a computer-aided on-line monitoring system
was established and applied for plant cell bioprocesses of
P. frutescens, and using this system, a detailed analysis of
the response of the cells’ physiological and metabolic
aspects such as cell growth and respiratory activity under
different agitation speeds was successfully conducted
(Zhong et al. 1994b).
Recently, flow cytometry has become a popular method
for ploidy screening, detection of mixoploidy and
aneuploidy, cell cycle analysis, and estimation of absolute
DNA amounts and genome sizes. Hence, it could be used
for biomonitoring plant cell-based processes (Yanpaisan et
al. 1999; Haas et al. 2008). The cell cycle is the process
whereby cells’ masses increase, their DNA content is
duplicated, their chromosomes segregate, and they divide
into daughter cells. Generally, the cell cycle can be divided
into four phases: the interphase or preparation phase
(designated the G1-phase), the DNA synthesis phase (S-
phase), followed by the postsynthetic gap phase (G2-phase),
and the mitosis or M-phase (Yanpaisan et al. 1999). As
shown in Fig. 4b, during batch processes, the cells progress
through these phases. A good relationship (r2 0.87)
between increases in cell weight and the proportions of
cells in the G0 +G1-phase can be observed between the first
and the tenth day of cultivation (Fig. 4c), demonstrating the
possibility of applying cell cycle kinetics measurements for
biomonitoring purposes as well as for synchronization of
the cell producers. However, it should be noted that the
G0 + G1 population may contain quiescent cells, thus, the
development of biparametric cell cycle analysis techniques
is essential (Haas et al. 2008).
Exhaust gas analysis provides further on-line parameters
that can be correlated to biomass concentration. Raval et al.
(2003) correlated the total oxygen consumption of an A.
indica suspension culture (measured by the shaken biore-
actor RAMOS) with the cell mass with an accuracy of ca.
80%. This type of correlation might be of special interest
for the cultivation of differentiated plant tissue, where
representative sampling is almost impossible, but exhaust
gas data are readily available.
819
Downstream processing
Although material from plant cell cultures is relatively
simple, compared to whole plants, it is still a complex
matrix. The pecto-cellulosic tissue and the membranes
enclosing organelles within cells represent significant
barriers for secondary metabolites to cross before they can
be released into a liquid medium. Thus, once a species has
been chosen and a strain selected to produce the target
metabolite(s) at high levels, or at least relatively high levels,
the extraction process has to be carefully chosen to
optimize primary recovery. Once in solution, the mixture
of secondary metabolites has to be purified, since only one
or few molecules are usually of interest. Most care has to be
taken in early steps of the purification process, when losses
of the metabolite(s) may be important. The scale of the
process (amount of pure metabolite expected) is a key
determinant of appropriate strategies to apply and the
required tools. The process should be as simple as possible,
especially when a scale-up will follow, and a model-based
approach may help to identify ways to increase the overall
efficiency (Degerman et al. 2008). The following data are
intended to give a practical overview of the steps involved
in the downstream processing of tissue cultures at lab and
pilot scales, from a natural product chemistry perspective,
focusing especially on processes involving liquid phases.
Extraction The optimal processes and conditions for
extracting targeted metabolites depend on their physico-
chemical nature and their intended use. For example,
alkaloids are ionizable molecules, so the pH of the
extraction medium is highly important. Furthermore, the
pH affects the stability of some compounds, e.g., antho-
cyanins, while stilbenes are prone to photocyclization and
may require protection from light. The metabolite’s polarity
may also influence the choice of the extraction solvent; e.g.,
Echinacea alkamides can be extracted by organic solvents
of low polarity or a water/ethanol mixture with a high
percentage of ethanol; whereas, polysaccharides can be
extracted by water. Use of solvents that may cause
interactions leading to structural changes, like alkaloids
with chloroform or electrophilic groups with methanol,
must be avoided (Maltese et al. 2009). The intended use of
the isolated metabolite should also be considered since
some solvents are forbidden or strictly regulated in food or
medicine processing. For example, Directive 88/344/CEE
regulates solvents that can be used in food and nutraceutical
production in the EU (Celex number 52008PC0154 at
http://eur-lex.europa.eu).
Metabolites of interest can be extracted from fresh or
dried material, in batch, semi-continuous or continuous
modes (although continuous extraction requires specific
instruments, several types of which are available). General
820
extraction procedures have been reviewed elsewhere re-
cently, by Romanik et al. (2007), and typical procedures for
cell cultures will be outlined here.
Cell walls can be ruptured before sample extraction by
homogenization, sonication, or steam explosion (Kurosumi
et al. 2006; Hartonen et al. 2007). The pretreatment
efficiency depends on the type of tissue extracted and the
metabolite localization, so ideally, any considered proce-
dure should be tested on a sample of the material to be
extracted. The selection and optimization of extraction
conditions is of importance. For example, optimization of
the ethanol proportion, temperature, pH, solid to liquid
ratio, and duration increased the yield of glucoraphanine in
batch extraction from Cardaria draba, and use of semi-
continuous extraction allowed an additional 50% yield
increase in experiments reported by Powell et al. (2005a, b).
A metabolite can be extracted from fresh homogenized
material with water or buffer, if it is soluble in an aqueous
medium, or with polar organic solvents (methanol or
ethanol), which simultaneously deactivate enzymes.
Since water is not a convenient solvent due to its high
boiling point (Bp), aqueous filtrates can be subjected to
liquid-liquid extraction, but such steps may lead to the
formation of recalcitrant emulsions. Commonly used
solvents include methyl or ethyl acetate (Bp 57°C and 77°C,
respectively), methyl tert-butyl ether (Bp 55°C), 1- or 2-
butanol (Bp 118°C and 100°C, respectively), and 2-methyl
tetrahydrofuran (Bp 80°C). Solvents like benzene, hexane,
and chloroform that are inherently toxic should be avoided,
and can be substituted, to some extent, by cyclohexane,
heptane, toluene, and methylene chloride. Oils and alkanes
(coconut oil or dodecane) can be used as extractive solvents
for lipophilic compounds such as carotenoids and com-
pounds of intermediate polarity such as scopoletin, and their
low toxicity may allow continuous extraction from living
cultures (Iizuka et al. 2005; Kang and Sim 2007, 2008).
Aqueous two-phase systems are usually used for protein
extraction, but are also starting to be used for low molecular
weight molecules like anthocyanins (Edahiro et al. 2005) or
even lipophilic molecules like lutein (Benavides et al. 2008).
Processes involving hydrophobic adsorption or ion-exchange
resins may also play useful roles in early processing stages,
to concentrate the metabolites of interest from the aqueous
medium (Silveira et al. 2008; Singh et al. 2009). For
instance, although they are best for trapping hydrophobic
compounds (Huang et al. 2008), glycosides like saponins
and iridoids can be successfully concentrated by hydropho-
bic resins.
Alternatively, biomass can be extracted in a dried state,
obtained through heating, or freeze-drying. Dried, ground
biomass can be directly extracted with organic solvents
depending on the polarity of the metabolite(s) of interest.
Lab-scale extraction can be performed by Soxhlet, reflux
Appl Microbiol Biotechnol (2009) 83:809–823
decoction under stirring or microwave heating (Mandal et
al. 2007). Alternatively, cold extraction methods may be
preferred, e.g., percolation, maceration, or sonication-
assisted maceration under stirring. The most efficient
physical treatment to extract metabolites from a natural
matrix is pressure (up to 100 bars), which forces solvent to
permeate the tissue thoroughly (Xie et al. 2009). However,
pressurized extraction requires purpose-built stainless steel
material and hardware, which is commercially available for
lab-scale extraction. Another technique that requires special
equipment, and is mainly applicable to apolar compounds,
is supercritical extraction (Reverchon and De Marco 2006).
Purification and isolation An interesting coincidence is
that the initial, pioneering work on chromatography was
performed at the same time as Haberlandt’s pioneering
plant culture trials (1902–1903). Although chromatography
is an elaborate and costly process, usually leading to losses
of compounds (especially when polar metabolites are
separated on silica), it can rarely be avoided. One of the
advantages of metabolite production by plant cell cultures
is that natural pathways favor one stereoisomer when chiral
structures are targeted. Nevertheless, closely related analogs
are usually produced besides the target metabolite and
require separation. Preparative chromatography can be
considered after preliminary purification of the sample (as
described above), and it should be applied to a fraction
enriched in the target metabolite. Diverse stationary phases
have been developed, allowing a wide array of applications,
and valuable books on its use are available (Hostettmann et
al. 1998; Sarker et al. 2005; Schmidt-Traub 2005).
Countercurrent chromatography is a technique of choice
for preparative chromatography applied to natural products,
regardless of the scale of the purification (Pauli et al. 2008).
Its interest lies in the fact that the mobile and stationary
phases are constituted by the two phases of a biphasic
solvent mixture. The liquid nature of the stationary phase
provides several advantages: there is no irreversible
adsorption, and the integrity of the sample can be
recovered; the choice of phases and thus selectivity is
virtually unlimited; the stationary phase has a considerable
loading capacity (limited only by the solubility of the
sample); it can be easily operated in displacement mode
(see below); phases are cheap (i.e., solvents); and overall
solvent consumption is significantly lower than in solid
phase chromatography. Countercurrent chromatography can
be applied under elution mode, in which the choice of
solvents can be guided either by the solubility of the sample
or by pre-designed solvent ranges (Renault et al. 2002;
Pauli et al. 2008). It can also be run (as mentioned) in
displacement mode, in which the addition of a displacer is
required to elute analytes (by switching the ionization state
of analytes or exchanging ionic pairs), and the analytes are
Appl Microbiol Biotechnol (2009) 83:809–823
separated according to their pKa or dissociation constant.
This mode is particularly suitable for preparative separa-
tion, since it allows high sample loads to be applied and
provides excellent separation. However, its utility is limited
by the need for the analytes to be ionized or ionizable.
Solvents and conditions are usually chosen according to the
results of a limited number of partition trials (Ito 2005).
Displacement countercurrent chromatography has been
applied to the preparative purification of rosmarinic acid
from Lavandula culture biomass (Maciuk et al. 2005),
huperzine A and B (Toribio et al. 2007a), and glucosino-
lates (Toribio et al. 2007b). Industrial-scale prototype
instruments are currently emerging from several manufac-
turers and are likely to gain increasing acceptance for
downstream processing.
Conclusion and future perspectives
Although commercial systems for cultivating plant cell
cultures were established more than 25 years ago, there has
been little progress towards wide application, and only a
limited number of compounds are produced at large-scale
today. This is due to several shortcomings of plant cell
cultures (notably the low yields of desired metabolites, their
biochemical and genetic instability, and several difficulties
resulting from the scale-up) and the availability of long-
established, well-accepted technologies for producing de-
sired compounds. However, the over-utilization of some
high-value plant species has led to serious ecological
problems and severe constraints of any further harvesting.
Hence, there is increasing interest once again in econom-
ically viable and eco-friendly technologies. Further, signif-
icant progress has been made in recent years towards the
development of low-cost, highly efficient and safe bioreac-
tor configurations, which could easily fulfill cGMP require-
ments. To maximize cost-effectiveness, the bioreactor’s
internal environment should be optimized, its operation
modes (fed-batch, continuous, and perfusion) should be
considered in detail, and the key parameters should be
determined. There have also been substantial advances in
downstream processing, and the application of industrial-
scale countercurrent chromatography could significantly
enhance overall cost-effectiveness. Although several prob-
lems still have to be addressed (e.g., better exploitation of
cells’ biochemical machinery and management of the
biosynthetic process is required), the progress made and
the identified tasks should lead to wide-scale commercial-
ization of plant cell cultures and contribute to the
preservation of global biodiversity.
Acknowledgements We thank Dr. V. Georgiev for preparing Fig. 1,
and Profs. J.J. Zhong (Shanghai Jiao Tong University), Y. Xu, and X.
821
Qian (East China University of Science and Technology, Shanghai,
China) for providing us with the relative prices of the novel elicitors
synthesized and used in their laboratories. Special thanks goes to D.
Ullisch, R. Huber, and Prof. Dr. J. Büchs (chair of Biochemical
Engineering, Aachen University of Technology, Germany) for
providing us with the MTP cultivations data. This work has been
supported by a National Science Fund of Bulgaria under contract
number DO 02-261/2008.
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