Fermentation Pilot Plant 41
3.0 BIOREACTORS FOR PLANT CELL TISSUE AND ORGAN
CULTURES fly Shinsaku Takayama)
3.1 Background of the Technique-Historical Overview
HaberlandtL'] first reported plant cell, tissue, and organ cultures in
1902. He separated plant tissues and attempted to grow them in a simple
nutrient medium. He was able to maintain these cells in a culture medium for
20 to 27 days. Although these cells increased eleven-fold in the best case, no
cell division was observed. GautheretL2] was the first to succeed in multiply-
ing the cells from the culture in 1934. He used the cambial tissues of her
pseudoplatanus, Salix capraea, Sambucus nigra. After 15 to 18 months in
subculture, cell activity ceased. He reasoned that this inactiveness was due
to the lack of essential substances for cell division. He suspected that auxin
may have been one of the deficient substances. This compound was first
reported in 1928 and was isolated by Kogel in the 1930's. Addition of auxin
to the medium prompted plant cell growth. This finding was reported almost
simultaneously by Gauthered3] and in 1939. Plant cell tissue and
organ culture techniques rapidly developed, and in the mid-1950's another
important phytohormone, cytokinins, had been discovered (Miller, Skoog,
Okumura, Von Saltza and Strong 1955).15] By 1962 Murashigeand Skoog[6]
had reported a completely defined medium which allowed the culture of most
plant cells. Their medium has now become the mostly widely used medium
in laboratories around the world.
After these initial discoveries and some significant improvements in
media, scientific research on the cultivation of plant cell, tissue, and organs
shifted to the area of basic physiological research. Industrial applications
were also sought in the production of secondary metabolites, clonal plants,
and the improvement of various plant tissues.
Plant cell, tissue, and organ culture can be performed by either solid or
liquid culture methods, however, in order to scale up the culture to the level
of industrial processes, the liquid culture method must be employed.
Recently, pilot bioreactors as large as 20 kl have been constructed in
the research laboratories of Japan Tobacco and Salt Co. and in those ofNitto
Denko Co. Solid culture methods were used in large scale pilot experiments
for the production of tobacco cells, and liquid culture methods were used in
the production of Panax ginseng cells. An outstanding example of cell
suspension culture in a pilot scale bioreactor (750 1) was the production of
shikonins by Mitui Petrochemical Industries. In all these examples, various
technologies have been used to improve the productivity of the metabolites.
42 Fermentation and Biochemical Engineering Handbook
The technologies include: (i) selection of a high yielding cell strain, (ii)
screening of the optimum culture condition for metabolite production,
(iii) addition of precursor metabolites, (iv) immobilized cell culture, and
(v) differentiated tissue andor organ culture. The productivity of various
metabolites such as ginsenoside, anthraquinones, rosmalinic acid, shikonins,
ubiquinones, glutathione, tripdiolide, etc., reached or exceeded the amount
produced by intact plants. To date, the production costs remain very high
which is why most of the metabolites are still not produced on an industrial
or pilot plant scale. Development oflarge scale industrial culture systems and
techniques for plant cell, tissue, and organs, and the selection of the target
metabolites are the chief prerequisites for the establishment of the industrial
production of plant metabolites.
I Culture Collection I
I I
Cell Culture
Figure 17. The area of plant cell, tissue and organ cultures.
Fermentation Pilot Plant 43
3.2 Media Formulations
The formulation ofthe medium for plant cell, tissue, and organ culture
depend primarily on nutritional requirements. Intact plants grow photoau-
totrophically in the soil, (i.e., they use CO, as the principal carbon source and
synthesize sugars by photosynthesis). In the case ofaseptic cultures however,
establishment of an autotrophic culture is not achieved so that heterotrophic
or mixotrophic growth becomes the distinguishing characteristic. Therefore,
such cultures require the addition of carbon as an energy source. Given this
fact, the culture medium must be formulated as a chemically defined mixture
of mineral salts (macro- and microelements) in combination with a carbon
source (usually sucrose). In addition to these constituents, organic constitu-
ents such as vitamins, amino acids, sugar alcohols, and plant growth
regulators are usually added to the medium. Media commonly used are listed
in Table 11.
Table 11. Formulations of most frequently used plant tissue
culture media
Ingredients (mg C1) MS B5 White Heller
(NH4)2s04
W4W3
KNo3
NaNO,
Ca(N03)2
CaCI,*2H20
MgS04*7H20
Na2SO4
=32po4
NaH,P O4.H2O
KCI
FeS O4.7H2O
Na,,EDTA
134
1650
1900 2500 80
300
440 150
370 250 720
200
170
150 16.5
65
27.8 27.8
37.3 37.3
600
75
250
125
75 0
(Cont’d next page)
44 Fermentation and Biochemical Engineering Handbook
Table 11. (Cont'd.) Formulations of most frequently used plant tissue
culture media.
Ingredients (mg 6-l) MS B5 White Heiler
FeCl3*6H2O
Mnso4-4H20
MnS04.H20
ZnS04.7H,0
K1
N%Mo04.2H20
CuS04.5H20
CoC12.6H20
NiC12*6H20
AlCI,
Myo-inositol
Nicotinic acid
P yridoxine.HC1
Thiamine.HC1
Glycine
Ca D-pantothenic acid
Sucrose
Kinetin
Fe2(S04)3
H3BO3
2,4-D
IAA
PH
22.3
8.6
6.2
0.83
0.25
0,025
0.025
100
0.5
0.5
0.1
2.0
30,000
0.04- 10
1.0-30
5.7-5.8
10
2
3
0.75
0.25
0.025
0.025
100
1 .o
1 .o
10.0
20,000
0.1
0.1-1 .o
5.5
2.5
7
3
1.5
0.75
0.5
0.1
0.1
3 .O
1 .o
20,000
6.0
5.5
1 .o
0.01
1
1
0.01
0.03
0.03
0.03
1.0
20,000
Fermentation Pilot Plant 45
3.3 General Applications
The most important fields of research for industrial applications, plant
cell tissue and organ cultures are clonal propagation and secondary metabo-
lite production. Plants cultivated in vitro have great changes in their
morphological features, from cell tissue to differentiated embryo, roots,
shoots or plantlets.
Applications to Secondary Metabolite Production. Plant tissue
culture is a potential method for producing secondary metabolites. Both
shikonins (Fujita and Tabata 1987)r'I and ginseng saponins (Ushiyama et al.,
1986)[*] have now been produced on a large scale by this method. However,
the important secondary metabolites are usually produced by callus or cell
suspension culture techniques. The amounts of some metabolites in the cell
have exceeded the amounts of metabolites in the cells of the original plants
grown in the soil. So it is expected that cell culturing may be applicable to
industrial processes for the production of useful secondary metabolites. It is
common knowledge that when a cell culture is initiated and then transferred,
the productivity of the metabolite decreases (Kurz and Constabel, 1979).['1
Once productivity decreases, it becomes very difficult to arrest or reverse the
decrease. In order to avoid this phenomenon, many cell strains were screened
to select those which would maintain metabolite productivity. Some metabo-
lites such as anthocyanins, shikonins, vinca alkaloids, and ubiquinones have
been reported to have increased their productivity significantly. Deus-
Neumann and Zenk ( 1984)[1°1 have checked the stability of the productivity
of the selected cell strains reported in the literature and noted that the
production of some metabolites such as anthraquinone (Uorinda citrofoliu),
rosmalinic acid (Colius blumei), visnagin (Ammi visnaga), diosgenin
(Dioscoreu deltoidea), etc., were stable after several subcultures, but some
metabolites such as nicotine (Nicotiana rusticu), shikonin (Lithospermum
erythrorhizon), ajmalicine (Catharanthus roseus), rotenoids (Derris eliptica),
anthocyan (Duucus carota), etc., were shown to be unstable after several
subcultures.
Clonal Plant Propagation. Plants are propagated clonally from
vegetative tissue or organs via bypass sex. Conventional clonal propagation
can be performed by leaf or stem cutting and layering or dividing ofthe plants,
however the efficiency is very low. Recently, many plants were propagated
efficiently through tissue culture. This technique was first reported in 1960
by G. Morel["] for the propagation of orchids and since then, many plants
have been propagated by tissue culture. Today there are many commercial
46 Fermentation and Biochemical Engineering Handbook
tissue culture nurseries throughout the world. Most of these tissue culture
nurseries are using flasks or bottles containing agar medium for commercial
propagation, but the efficiency is also low. In order to improve the efficiency,
use of a bioreactor is desirable. Using a small bioreactor (4 to 10 liters), the
author has produced over 4,000 to 10,000 plantlets within 1 to 2 months. The
bioreactor system allows the induction of somatic embryos from vegetative
cells which then leads to the production of artificial seeds (Redenbaugh et al.,
1987).['*]
3.4 Bioreactors-Hardware Configuration
The configuration of bioreactors most frequently used for plant cell,
tissue, and organ cultures is hndamentally the same as that used for microbial
or animal cell cultures. However, in plants, the cells, tissues, and organs are
all susceptible to mechanical stresses by medium aeration and agitation. At
times, the production of both cells mass and metabolites is repressed severely
and the bioreactor must therefore have the characteristics of low shear
stresses and efficient oxygen supply. For these reasons, different bioreactors
(Fig. 18) have been investigated in order to select the most suitable design.
Wagner and Vogelmann ( 1977)[131 have studied the comparison of different
types of bioreactors for the yield and productivity ofcell mass and anthraqui-
none (Fig. 19). Among different types of bioreactors, the yield of anthraqui-
nones in the air-lift bioreactor was about double that found in those
bioreactors with flat blade turbine impellers, perforated disk impellers, or
draft tube bioreactors with Kaplan turbine impellers. It was also about 30%
higher than that of a shake flask culture. Thus, the configuration of the
bioreactor is very important and development efforts are underway for both
bench scale and pilot scale bioreactors.
Aeration-Agitation Bioreactor. This type of bioreactor (Fig. 20) is
popular and is fundamentally the same as that used with microbial cultures.
For small scale experiments, the aeration-agitation type bioreactors is widely
used. However, when the culture volume is increased, many problems arise.
The following are some of the scale-up problems in large aeration-agitation
bioreactors: (i) increasing mechanical stresses by impeller agitation and (ii)
increasing foaming and adhesion of cells on the inner surface of the
bioreactor. Despite these problems, a large scale pilot bioreactor (volume 20
kl) was constructed. It successfully produced both cell mass and metabolites.
This bioreactor is therefore the most important type for bioreactor systems.
Fermentation
Pilot
Plant
47
48 Fermentation and Biochemical Engineering Handbook
10 I
0.5 wrn
100 rprn
I
101
0.5 wm
100 rprn
I
u4
75 I
0.33 vvm
350 rprn
dry weight metabolite JJ dry weight metabolite
yield productivity
Figure 19. Comparison of yield and productivity for cell mass and anthraquinones in
various bioreactor systems. (1) Shake Flask. (2) Flat Blade Turbine. (3) Perfolated Disk
Impeller. (4) Draft Tube Bioreactor with Kaplan Turbine. (5) Air-lift Bioreactor.
Air Driven Bioreactors. The simplest design is the air-driven
bioreactor equipped with sparger at the bottom ofthe vessel. It is widely used
for plant cell, tissue, and organ cultures. In cases where the cells grow rapidly
and the cell mass occupies 40-60% of the reactor volume, the flow charac-
teristics become non-Newtonian and the culture medium can no longer be
agitated by simple aeration,
Rotating Drum Bioreactor. The rotating drum bioreactor (Fig. 21)
turns on rollers and the oxygen supply mechanism is entirely different from
either the mechanically agitated or the air-lift bioreactor. Tanaka et al.,
( 1983),[14] reported that the oxygen transfer coefficient is affected by a
change of airflow rate under all rotational speeds (Fig. 22). This character-
istic is suitable not only for thegrowth ofplant cell, tissue, and organs but also
for the production of metabolites under high viscosity and high density
cultures. It is superior to the cultures using either mechanically agitated or
air-lift bioreactors since the cultures are supplied ample oxygen and are only
weakly stressed. Recently a 1 kl bioreactor of this type was constructed and
used for a pilot scale experiment (Tanaka 1987).[15]
49Fermentation Pilot Plant
Figure 20. Ninety-five liter automated bioreactor for plant cell, tissue and organ cultures.
(photo courtesy ofK. F. Engineering Co., Ltd., Tokyo).
BAFFLE OXYGEN
PLATE SENSOR
￥
~8AIR-~
(!)
Figure 21. Schematic diagram of the rotating drum bioreactor (Tanaka, H., et al., 1983)
Ro!ation speed
5:: 0 AIR FLOW RATE (wm) 5
Figure 22. Effect ofthe airflow rate on k,a in rotating drum fermenter. (Tanaka, H., et al.,
1983)
Spin Filter Bioreactor. This type of bioreactor (Styer, 1985)[161 is
equipped with a filter driven by amagnetic coupling in the stir plate (Fig. 26).
This spinning filter operates as a medium agitator without generating shear
stress and also serves as an excellent filter for the removal ofthe medium from
the bioreactor without the cells plugging it. The spin filter bioreactor will be
most suitable for the continuous culture of plant cells. When a conventional
bioreactor was used and the feeding rate ofthe medium was increased, the cell
density was decreased because of washout. However, when a spin filter
bioreactor was used, the cell density was maintained constant and half of the
spent medium was effectively removed through the spin filter.
Gaseous Phase Bioreactor. As shown in Fig. 24, this type of
bioreactor is equipped with filters on which the culture is supported and with
a shower nozzle for spraying on the medium (Ushiyama et al., 1984;[”]
Ushiyama, 1988).[’*] Seed cultures are inoculated on the filters and the
medium is supplied to the culture by spraying from a shower nozzle. The
drained medium is collected on the bottom of the bioreactor. This type of
bioreactor is excellent for plant cell, tissue, and organ cultures because there
is no mechanical agitation (e.g., driven impeller, aerator) and, therefore, the
growth rate and the secondary metabolite production are enhanced.
Light Introducing Bioreactor. Plants are susceptible to light
irradiation and as a consequence various metabolic and/or physiological
changes are generated. Some important reactions are: (i) photosynthesis,
(ii) activation of specific enzymes such as phenylalanine ammonia lyase
(PAL) and to induce the production of flavonoids or anthodyanins, (iii)
photomorphogenesis such as development of leaves. For these reactions, the
Fermentation Pilot Plant 51
introduction of light into the bioreactor is required. Inoue ( 1984)[191 reported
a bioreactor equipped with transparent pipes. The light was emitted from the
surface of the pipe into the bioreactor. Ikeda ( 1985)[20] reported an air-lift
bioreactor equipped with a photo introducing draft tube (Fig. 25). The draft
tube was constructed as an airtight tube which consisted of a transparent
inner and outer tube. Within the center of the draft tube was a light
introducing optical fiber. The light source was a sunlight collector system
which operated automatically by computer control and the collected light
was introduced into the bioreactor through the optical fibers. Introduction
of light into the bioreactor will become an important technique for the
production of specific plant metabolites.
SPENT
MEDIUM
MEDIUM
SPIN -
F I LTE RI
+ SPENT MEDIUM
AND CELLS
- SPARGER
Figure 23. The spin filter bioreactor (Styer, 1985).
52 Fermentation and Biochemical Engineering Handbook
17
nl
12
I00
7131 ./ +I 01
I01
12
IO 161
1
Figure 24. Gas phase bioreactor (Ushiyama, et al., 1984).
Fermentation Pilot Plant 53
n
“1 1
Figure 25. Photo introducing bioreactor (Ikeda, 1985).
54 Fermentation and Biochemical Engineering Handbook
Gas Permeable Membrane Aerator Bioreactor. This type of
bioreactor has not yet been fully developed. Nevertheless, some information
is available. For example, one bioreactor is equipped with an aerator
composed of fine tubes made of polycarbonate, polypropylene, silicone gum,
etc. This type of bioreactor should be valuable for immobilized plant cell
cultures.
3.5 Bioreactor Size
For industrial production of secondary metabolites, large scale biore-
actor systems (sometime over 100 kl) will be required. The 75 kl and 20 kl
bioreactor systems used for pilot scale experimentation are at present the
largest in the world. They are at the DIVERSA Gesellschaft fiir Bio- und
Verfahlenstechnik mbH in Germany and Nitto Denko Co., Ltd. in Japan.
When there is a limited demand for a particular metabolite (e.g., a pigment),
the production of commercial quantities can be done in the pilot scale
bioreactor. Shikonin is produced this way. In 1983, Mitui Petrochemical
Industries became the first to commercially produce a plant metabolite by
using a 750 1 bioreactor. For routine experiments, the smaller bioreactors of
vessel volume 1 to 100 liters are more widely used. Small bioreactors with
volumes from 1 to 20 liters are used commercially for the production of clonal
plants. These small bioreactors are valuable for the rapid propagation of
large numbers of clonal plantlets. Through asexual embryogenesis, 10,000
to 1,000,000 embryos can be produced per liter and these embryos are then
grown to plantlets. Using 2 to 10 1 bioreactors, it is also possible to produce
5,000 to 10,000 plantlets from plant tissue, which can then be transplanted
directly into soil.
3.6 Culture Period
The growth of plant cells, tissues, and organs is much slower than
microbial organisms. The most rapid growth cell line reported in scientific
journals is the bright yellow Nicotiana tabacum cv. (Noguchi et al.,
1987).[211 The doubling time ofthis cell strain was 15 h, and the duration to
maximum growth was 80 h (3.3 days) when cultured in a 20 kl pilot scale
bioreactor. In general, the growth of the cells of herbaceous annual plants
is rapid and their doubling time is usually about 1 to 3 days (duration to
maximum growth was 10 to 20 days), and that of woody plants or
differentiated organs is slow (doubling time is about 2 to 10 days and the
culture period is about 20 to 100 days).
Fermentation Pilot Plant 55
3.7 Aeration and Agitation
The oxygen requirement of plant cells is quite low compared to
microorganisms. Kato et al. (1975)r2*1 have examined the effect of k,a on
biomass production (Fig. 26a). They observed that the volumetric oxygen
transfer coefficient, $a was constant after 10 h and the final biomass
concentration became constant at 0.43 g cell dry weight/g sucrose. Whenk,a
was set under 10 h, cell yield became dependent on k,a values. The effect of
agitation speed on final cell mass concentration was also analyzed by Kato
et al. ( 1975)[221 using a 15 1 bioreactor (Fig. 26b). At lower agitation speeds
(less than 150 rpm), cell mass concentration became constant. However, when
the agitation speed exceeded 150 rpm, the cultures became bulky and started
to foam profusely. An agitation speed of either 50 or 100 rpm seemed to be
optimal for production of cell mass and also for avoiding the culture problems.
201 I I I I I
h
-
.
15 1
I I I 1 I
0 5 10 15 20 25 30
P P
I
15-
5
10-
5-
0 I I I I I
100 150 200 0 50
AGITATION SPEED ( rprn )
Figure 26. Effect of initial oxygen transfer coefficient and agitation by turbine impeller on
cell growth (Kato, 1975).
56 Fermentation and Biochemical Engineering Handbook
3.8 Microbial Contamination
According to Manfredini et al. (1982),[231 the most frequent factors
causing microbial contamination include: (i) construction materials, (ii) seals
and valves, (iii) complexity of the plant, (iv) operator error, (v) instrumen-
tation failure, (vi) process air, (vii) transfer and feed lines, (viii) contamina-
tion from vegetative preculture, (ix) critical medium composition, (x) inad-
equate procedures. As the culture periods of plant cell, tissues, and organs
are usually quite long, (particularly for continuous cultures), special designs
and operations are necessary to avoid microbial contamination. For example,
Hasimoto et al. ( 1982),[241 reduced contamination in their 20 kl bioreactor by
using three air filters in a series; the third filter was a membrane of uniform
pore size of 0.4 pm. The design specified aseptic seals in the agitator shaft
and exit pipelines for sterilized air after steam sterilization.
3.9 Characteristics
The special characteristics of plant cells that tend to hamper large
scale cultivation of the cells are described below.
Bubbling and Adhesion of Cells to the Inner Surface of the
Bioreactor. Plant cell cultivation is usually performed by bubbling and
agitation which cause foaming and adhesion of the cells to the surface of a
bioreactor. Because of this phenomenon, cell growth is inhibited. The
authors (Takayama et al., 1977)[251 have examined the possible causes and
concluded that the adhesion of cells appeared to be the result of gel formation
from pectin and calcium. By reducing the concentration of CaC12-2H20 in
the medium, the foaming and the number of cells that adhered to the walls was
decreased markedly. The cells became easily removable from the inside wall
of the fermenter and were returned to the medium. Cell destruction was
measured by A660 values which also depend on the CaC12.2H20 levels. A
lower level of CaC12.2H20 in the medium markedly inhibits cell destruction.
These observations are particularly pertinent when large-scale cultivation is
being considered.
According to Tanaka
(1 982),[26] plant cells have a tendency to grow in aggregates of different sizes.
The size distribution of cell aggregates is different from one plant species to
another (Tanaka, 1982).[27] Specificgravity ofthese cells ranges from 1.002
to 1.028. Ifthe diameter ofthe cell aggregate is less than 1 to 2 mm, the cells
can be suspended and do not sink to the bottom of the bioreactor (Tanaka,
1982),[261 but, when the specific gravity of the cell is greater than 1.03, the
Cell Morphology and Specific Gravity.
Fermentation Pilot Plant 57
diameter of the aggregate becomes 0.5 to 1.0 cm and the cells sink to the
bottom of the bioreactor and cannot be suspended. When the agitation is
increased, the size of the cell aggregate becomes smaller (Tanaka, 198
but the growth of the cell is repressed. In order to separate the cells from the
aggregate, the amount of calcium is decreased to suppress the gel formation
ofpectin which plays an important role in the cell, e.g., cementing plant cells,
but has little effect on cell separation (Takayama, 1 977).[251
Viscosity, Fluidity, and Oxygen Supply. When plant cellsgrow well,
they can occupy 40 to 60% of the whole culture volume, and the apparent
viscosity becomes very high. Tanaka ( 1982)[26] examined the relationship
between apparent viscosity and concentration of solids in suspension, and
concluded that when the cell density exceeds 10 g/l, the slope of the apparent
viscosity increases rapidly, and when cell density reaches 30 gA, the culture
medium becomes difficult to agitate and supply with oxygen.
100
80 -
60 -
40-
-
d
ir
Y
E 20-
$
E 10-
>
2 6-
4-
I- 8-
$
a
1I-
II I It I I I
4 6 810 20 40 60
CONCENTRATION ( g cell I I )
Figure 27. Relationship between apparent viscosity and concentration of cells and
pseudocells in culture media (Tanaka, 1982). (0) C. Roseus, (0) C. Tn'cupsidata B.,
(A)N tabacum L., (+) granulated sugar.
58 Fermentation and Biochemical Engineering Handbook
3.10 Manipulation
Large Scale Batch Culture. Batch culture systems are in use
worldwide and many experimental results have been reported using 10 1 to 20
kl bioreactors. Noguchi et al. (1 987)L2'1 have examined the growth oftobacco
BY-2 cells using a 20 kl aeration-agitation bioreactor with 15 kl medium. The
medium used was Murashige and Skoog's inorganic nutrients with three
times the normal amount ofphosphate and 3% sucrose, incubated at 28OC and
aerated at 0.3 wm. The results revealed that the highest growth rate was
observed from the incubation time of 45 to 70 hours with a doubling time of
about 15 hours, which was almost the same as the growth in flask cultures.
Ushiyama et al. (1 986)['l examinedthe growth of Panax ginseng root cultures
in 30 1,2 kl, and 20 kl aeration-agitation bioreactors. The productivity ofthe
cultures in 2 kl and 20 kl bioreactors was 700 and 500 mg/l/day in dry weight,
respectively. Building upon this basic research, large scale batch culture
techniques have been developed for the industrial production of cell mass.
However, culture conditions suitable for cell mass production are not always
suitable for secondary metabolite production. Accordingly, in order to
produce both cell mass and metabolites efficiently, two-stage culture tech-
niques have beenadopted. This technique uses two batch bioreactors and was
first reported by Noguchi et al. ( 1987)[21] for the production of low nitrogen
content tobacco cells. In the 1980's, this technique was widely used for
secondary metabolite production such as shikonin (Fujitaet al.),[27] rosmarinic
acid (Ulbrich, 1985)[*'1 and digoxin production (Reinhard, 1980).[281
For shikonin production by Lithospermum eryfhrorhizon, two-stage
cell culture was used (see Fig. 28). The first stage culture was grown in a MG-
5 medium which was suitable for cell mass production. It was then
transferred to 2nd-stage culture where it was grown in an M-9 medium,
modified by a higher Cutt content and a decreased salt content.
Large Scale Continuous Culture. The growth rate of plant cells is
usually low compared to that of microbial organisms. In order to enhance the
productivity of cell mass and metabolites, continuous culture methods should
be employed (Wilson, 1978;[291 Fig. 29a). In the research laboratories of
Japan Tobacco and Salt Co., a pilot plant (1500 1) and an industrial plant
system (20 kl) have been used for developing continuous culture techniques
(Hashimoto, et al., 1982;[241 Azechi et al., 1983r3O]). A 20 kl bioreactor
having a working volume of 6.34 kl, was used for the experiment (Fig. 29b)
and ran for 66 days of continuous operation. The conditions were: aeration
rate, 0.35-0.47 wm; agitation speed, 27.5-35 rpm; dilution rate, 0.28-0.38
Fermentation Pilot Plant 59
days. In this experiment, the residual sugar content was an important index
of the operation and, at steady state, its value was maintained above 5 gA.
Other control parameters such as aeration, agitation, and dilution rates were
changed gradually. The success of this experiment will soon lead to the
establishment of long term industrial continuous culture systems for second-
ary metabolite production.
Immobilized Culture. Immobilization ofplant cells was first reported
by Brodelius et al. in 1979,L3l1 and since then many reports have been
published. Unfortunately, an immobilized cell culture technique has not yet
been established as an industrial process for secondary metabolite produc-
tion. However, this technique has many excellent features and should be the
subject of fhture development research.
MEDIA
7-
CELL
M-9
MEDIA
2nd STAGE
CULTURE
I In
AIR SPENT AIR SPENT
MEDIUM MEDIUM
Figure 28. Two stage culture methods (Fujita, 1984).
60
Fermentation
and
Biochemical
Engineering
Handbook
A n -
m
Y
m
Fermentation pilot Plant 61
'0' ring seal
Inoculum Port
Hollow
fibres
culture space
Continuous feed
feed
I
Cell debris
Quiescent
Seconda Shell
plant
metabolit side
Porous
support
Lumen
side
Bacterial contaminants
Figure 30. Immobilized plant cell cultures. (Prenosil and Pederson 1983).
3.11 Scale-up Problems
Scale-up techniques for plant cell cultures are not well understood.
Some attempts have been made using tobacco cells and applying essentially
the same parameters as those for the scale-up of microbial cultures (Azechi,
1985).[33] The results showed that &u values are useful as scale-up
parameters, however, the situation for secondary metabolite production is
quite different. The productivity of the metabolites decreased as the culture
volume increased. An example of this is the productivity of the indole
alkaloid, a serpentine which declined significantly as the culture volume
increased from 0.1 to 80 1 (Fowler, 1987).[341 Possible reasons for the loss
of product on scale-up are the following: (i) altered and inadequate mixing
of the nutrient and cells at the high reactor volumes and (ii) lowered dissolved
oxygen level (Breuling et al, 1985).[351 Fujita and Tabata (1987)['] used the
scale-up of suspension cultures of Lithospermum erythrorhizon cells for
their ability to produce shikonins as the criterion for comparing the aeration-
agitation type bioreactor with a modified paddle impeller and the rotary drum
62 Fermentation and Biochemical Engineering Handbook
type bioreactor. When the aeration-agitation type bioreactor was scaled up
to a volume of 1000 1, the shikonin productivity decreased, but when the
rotary drum bioreactor was scaled up to 1000 1, there was no decrease in the
yield of shikonins (Fig. 3 1). Thus, in an industrial pilot plant for secondary
metabolite production, mild agitation and oxygen supply will be important
variables.
--
E ,OWt
I
v
1
1
Inoculum : 6 gDW / I
-G Rotary Drum
Type Tank
-0- Aeration -
Agitation Type
Tank with a
Modified Paddle
Impeller
I I I
10 100 1000
TANK VOLUME (I)
Figure31. Scale up of suspension cultures for Lithospennum erythrorhizon cells. (Fujita
and Tabata, 1987).
3.12 Bioprocess Measurement and Control
Bioprocess Measurement and Control in Large Scale Culture.
Measurement and control systems used in the bioreactor for plants are
essentially the same as those for microbial or animal cell cultures, but, in
special cases, where the mineral components influence the productivity of
secondary metabolites, the kind of salts used for the electrode must be taken
into consideration.
Mass balance. Themass balanceequation (Eq. 4) is generally used for
heterotrophic organisms. Pareilleus and Chaubet (1 980)[36] have applied the
equation to apple cell cultures:
1 dS 1
- * (P + m)
YG
Eq. (4) X’z =
Fermentation Pilot Plant 63
where
1 dS
dt
= specific rate of sugar consumption
(g sugar / g cell dry weight e h)
-.-
p = specific growth rate (h)
YG = maximum growth yield (g cell weight /g sugar)
m = maintenance coefficient for sugar (g sugar /g cell
dry weight/%)
From the equation, m and YG can be estimated from a plot of b". (dS/dt)
against p, The values observed for Y, and m are in good agreement with those
reported previously by Kat0 and Nagai ( 1979)[371 calculated from tobacco
cell cultures; i.e., m values were smaller and Y, values were higher compared
to the values reported for many microorganisms.
Measurement and Mathematical Fitting of Cell Growth. Richards
(1960) used a generalized logistic curve for the mathematical fitting of the
growth curve of plants. The equation is:
A-W
W-B
In = f (t)
where
A = asympotic value of dry weight
B = inoculated value of dry weight
t = days culture in vitro
W
=
dry weight at any instant t (days or hours)
f(t) = suitable function of time t (days or hours)
By eliminating the natural logarithm
Differentiating with respect to t:
64 Fermentation and Biochemical Engineering Handbook
The above equation is the growth rate. To calculate the specific growth
rate, u, divide by the dry weight
Doubling time, (T) becomes:
References (Section 3)
1. Haberlandt, G., Kulturversuche mit isolierten Pfanzen, Sber. Akad. Wiss.
Wen., 1 1 1 :69-92 (1 902)
2. Gautheret, R. J., Culture de tissue cambial. C. R. Hebd. Seanc. Acad. Sci.,
3. Gauthret, R. J., Sur la possibilite de realiser la culture in definie des tissu
detubericulede carotte. C. R. Hebd. Seanc. Acad. Sci., Paris, 208:118-121
(1939)
4. White, P. R. Potential unlimited growth of excised plant callus in an
artificial medium, Amer. J. Bot., 26:59-64 (1939)
5. Miller, C. O., Skoog, F., Okumura, F. S., Von Saltza, M. H., and Strong, F.
M., Structure and synthesis ofkinetin.,J. Amen'. Chem. Soc., 77:2662-2663
(1955)
6. Murashige, T. and Skoog, F., A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol. Plant, 15:473-497 (1962)
7. Fujita, Y. and Tabata, M., Secondary metabolites from plant cells-
Pharmaceutical applications and progress in commercial production, Plant
Tissue and Cell Culture, pp 169-185, Alan R. Liss Inc., (1987)
8. Ushiyama, K., large scale culture techniques ofplant cells and the secondary
metabolite production, Hakko to Kogyo, 46:7-11 (1988), in Japanese.
9. Kurz, W. G. W. and Constabel, F., Plant cell suspension cultures and their
biosynthetic potential, Microbial Technol., 1:389416 (1979)
10. Deus-Neumann, B. and Zenk, M. H., Instability of indole alkaloid produc-
tion in Catharanthus roseus cells in suspension cultures, Planta Med.,
50:42743 1 (1984)
Paris, 198:2195-2196 (1934)
Fermentafion Pilot Plant 65
11. Morel, G., Producing virus-free cymbidium, Amer. Orchid SOC. Bull.
29:495497 (1960)
13. Wagner, F. and Vogelmann, H., Cultivation of plant tissue culture in
bioreactors and formation of secondarymetabolites. (W. Ban, E. Reinhard,
M. H. Zenk, Eds.), Plant Tissue and Its Bio-technological Applications, pp.
245-252, Springer Verlag, Berlin, ISBN 3-540-08227-1, (1 987)
14. Tanaka, H., Nishijima, F., Suwa, M., and Iwamoto, T., Rotating drum
fermentor for plant cell suspension cultures, Biotechnol. Bioeng., 25:2359-
2370 (1 983)
15. Tanaka, H., Large-scale cultivation ofplant cells at high density: A Review,
Process Biochem, pp. 106-1 13 (1 987)
16. Styer, D. J. in: Bioreactor Technology forPlantPropagution, (R. R. Henke,
K. W. Hughes, M. J. Constantin, and A. Hollander, Eds.), pp. 117-130,
Plenum Press, ISBN 0-30641 91 9 (1 985)
17. Ushiyama, K., Oda, H., Miyamoto, Y., andIshida, Y., Japan Patent, Kokai,
5945879 (1984)
18. Ushiyama, K., Oda, H., and Miyamoto, Y., Large scale tissue culture of
Panax ginseng root, Abst. VIth International Congress ofplant Tissue and
Cell Culture, p. 252, University of Minnesota (1 986)
19. Inoue, H., Culture vessel for Photo-requiring organisms, Japan Patent,
Kokai 59-21682 (1984)
20. Ikeda, H., Culture vessel for photoautotrophic culture, Japan Patent, Kokai
21. Noguchi, M., Matumoto, T., Hirata, Y., Kamamoto, Y., Akutu, A,, Kato, A.,
Azechi,A., andKato,K.,Improvement ofgrowthratesofplantcell cultures.,
in: Plant Tissue Culture and Its Bio-technological Application, pp. 85-94,
Springer Verlag, Berlin, ISBN 3-540-18227-1 (1987)
22. Kato, A,, Shimizu, Y., and Noguchi, S., Effect of initial kLu on the growth
of tobacco cells in batch culture, J. Ferment. Technol. 53:744-75 1 (1 975)
23. Manfredini, R., Saporiti, L G., and Cavallera, V., Technological approach
to industrial fermentation: limiting factors and practical solutions, La
Chimica E I'Industria, 64:325-334 (1982)
24. Hashimoto, T., Azechi, S., Sugita, S., and Suzuki, K., Large scale production
oftobacco cells by continuous cultivation, Plant tissue Culture, pp. 403404
(1 982), Maruzen Co., Tokyo (1 982)
25. Takayama, S., Misawa, M., KO, K., and Misato, T., Effect of cultural
conditions on the growth ofAgrostemma githago cells in suspension culture
and the concomitant production of an anti-plant virus substance, Physiol.
Plant, 4 1:3 13-320 (1 977)
26. Tanaka, H., Some properties of pseudo cells of plant cells, Biotechnol.
Bioeng., 24:2591-2596 (1982)
27. Tanaka, H., Oxygen transfer in broth ofplant cells at high density,BiotechnoL
Bioeng., 24:425442 (1982)
28. Tanaka, H., Technological problems in cultivation of plant cells at high
density, Biotechnol. Bioeng., 23:1203-1218 (1981)
60-237984 (1985)
66 Fermentation and Biochemical Engineering Handbook
29. Wilson, G., Growth and product formation in large scale and continuous
culture systems, Frontiers ofplant Tissue Culture, pp. 169-1 77, University
of Calgary, Canada (1 978)
30. Azechi, S., Hashimoto, T., Yuyama, T., Nagatsuka, S., Nakashizuka, M.,
Nishiyama, T., and Murata, A., Continuous cultivation of tobacco plant cells
in an industrial scale plant, Hakkokogaku, 60:117-128 (1983)
31. Brodelius, P., Deus, B., Mosbach, K., and Zenk, M. H., Immobilized plant
cells for the production and transformation of natural products., FEBS
Letters, 103:93-97 (1979)
32. Prenosil, J. E. andpederson, H., Immunobilized plant cell reactors, Enzyme
Microtechnol., 5:323-331 (1983)
33. Azechi, S., Large scale culture of plant cells, in: Bio-Engineering, Nikkan
Kougyou Shinbunsya (1 985)
34. Plant Tissue and Cell Culture, (D. D. Blesboer, ed.), pp. 459-471 , Alan
R. Liss Inc., ISBN 0-8451-1802-1 (1987)
35. Breuling, M., Alfermann, A. W., and Reinhard, E., Cultivation of cell
cultures of Berberis wilsonde in 20-1 air-lift bioreactor, Plant Cell Report,
36. Pareilleux, A. and Chaubet, N., Growth kinetics of apple plant cell cultures,
Biotechnol. Letters, 2:29 1-296 (1 980)
37. Kato, A. and Nagai, S., Energetics of tobacco cells, Nicotiana tabacum L.
growing on sucrose medium, Europ. J. Appl. Microbiol. Biotechnol., 7:2 19-
255 (1979)
38. Fowler, M. H., Process systems and approaches for large scale plant cell
cultures, in: Green, C. E., Somers, D. A,, Hackett; W. P., and Fujita, Y,;
Shikonins, in: Plant Tissue Culture andFine Chemicals, pp 19 1-1 97, CMC
Press Inc., Tokyo (1984)
39. Redenbaugh, K., Viss, P., Slade, D., and Fujil, J A., Scale-up; artificikal
kalin, Plant Tissue and Cell Cultures, pp. 473-493 (1987)
40. Reinhard, E. and Alfermann, A. W., Biotransformating by plant cell
cultures, Adv. Biochern. Engineer, 16:49-83 (1980)
41. Tanaka, H., Nishijima, F., Suwa, M., and Iwamoto, T., Rotating drum
fermenter for plant cell suspension cultures, Biotechnol. Bioeng., 25:2359-
2370 (1983)
42. Ulbrich, B., Welsneer, W., and Arens, H., Large-scale production of
rosmarinic acid from plant cell cultures ofColeus blumei Benth in: Primary
and Secondary Metabolism ofPIan Cell Cultures, (K. H. Neumann et al.,
Eds.), pp. 293-303, Springer-Verlag, Berlin (1 985)
4:220-223 (1985)
