Plant Cell Reports
Plant Cell Reports (1986) 5:302-305
© Springer-Verlag1986
Alkaloid accumulation in Ca-alginate entrapped cells of Catharanthus roseus: Using a limiting growth medium Florence Majerus and Alain Pareilleux D6partement de G6nie Biochimique et Alimentaire, UA-CNRS-N°544, Institut National des Sciences Appliqu6es, Avenue de Rangueil, F- 31077 Toulouse C6dex, France Received May 22, 1986 / Revised version received June 20, 1986 - Communicated by A. M. Boudet
ABSTRACT
continued growth of cells. There is evidence that plant
The limitation of growth of Cathavanthue r o s e u s cells
secondary metabolites are often produced at higher
was investigated with a view to their entrapment in a
concentration in slow growing or non-growing cultures
Ca-alginate matrix. An examination of the effects of
(Lindsey and Yeoman, 1983). This is the case for
lowered 2,4-D and phosphate concentrations on cell
Oathavanthus r o s e u 8 cells in which alkaloid accumulatio~
v i a b i l i t y and indole alkaloid biosynthesis enabled a
is restricted to the post exponential growth phase
growth limiting and product formation stimulating
(Pareilleux and Vinas, 1982). Obviously, i f the biocata-
medium to be designed. Entrappedcells showed a
l y t i c capacity of the cells could be maintained in
retention of both respiratory activity and biosynthetic
growth limiting conditions, the h a l f - l i f e of immobilized
capacity over an extended period of time comparedwith
systems should be increased, and the production yield
free cells. Evidence is presented which suggests that
enhanced. In this study, we investigated the capability
immobilization in Ca-alginate beads acts to stabilize
of growth-limited, suspendedand immobilized cells of
cells, resulting in enhancedproduct accumulation.
Oatharanthus roseus
ABBREVIATIONS
2,4-D : 2,4-dichlorophenoxyacetic acid; d.w.: dry weight.
MATERIALS AND METHODS
Cell suspension cultures. Cell suspension cultures of Cathavanthus
INTRODUCTION
Plant cell cultures have great potential in biochemical production and biotransformation reactions. With a view
to synthesize indole alkaloids.
roseus
(L.) G Don were maintained and
subcultured every week in the medium described by Gamborg et al. (1968) supplementedwith 4.5 ~M 2,4-D and 0.28 ~M kinetin and the addition of 3 % sucrose as
to enlarge production increased productivity of useful
the carbone source (basal medium). The suspensions were
compounds may be obtained using a continuous mode of
cultivated in the dark at 27°C in 250 ml Erlenmeyer
operation working over long periods. There are a number
flasks with 100 ml mediumper flask. When otheK media
of advantagesassociated with the use of immobilized
were tested, the cells were harvested by f i l t r a t i o n ,
or sequestered cells ; for instance, dilution rates in
washed and resuspended in the appropriate medium. The
excess of the growth rate of the culture can be used. A variety of methods for plant cell immobilization
30 g/l in the basal medium. The composition of the
have now been reported including entrapment in a
limiting mediumwas the sameas the basal mediumexcept
2,4-D free medium contained 50 g/l sucrose instead of
polymeric matrix (Brodelius et a l . , 1979 ; Brodelius
the concentrations of 2,4-D, phosphate and CaCl2 which
and Nilsson, 1980 ; Galun et a l . , 1983), covalent
were changed (45 nM, 100 ~M and 5 mM r e s p e c t i v e l y ) . The
linkage to polyphenylene oxide (Jirku et a l . , 1981),
f r e e l y suspended c e l l cultures were started with an
adsorption to microporous polyurethane foam (Lindsey
inoculum of 0.1-0.2 g d.w. per f l a s k . In entrapped c e l l
et a l . , 1983), entrapment in hollow fiber reactors
experiments, Erlenmeyer flasks were inoculated with 10 g
(Shuler, 1981). Most commonly, c e l l s are entrapped in
of beads (0.2 g d.w. per f l a s k ) Immobilization of c e l l s . Immobilization in Ca-alginate
a gel matrix, usually calcium a l g i n a t e . However, one of the problems with plant c e l l s immobilized in Ca-
was performed under aseptic conditions by the follow.img
alginate is the i n s t a b i l i t y of the matrix, due to a
method : c e l l s of a 7 day-old suspension were f i l t e r e d
Offprint requests to: A. Pareilleux
303 through a 800 ~m metal net and retained on a 40 ~m net. 250 g wet weight of c e l l s were suspended in 300 ml of
biomass
(g/I)
(%)
viability
,= z~
00
3 % a l g i n a t e dissolved in the l i m i t i n g medium and the suspension was added dropwise to the medium containing
10.
50 mM CaCI2. The beads were l e f t in t h i s solution f o r I h with s t i r r i n g and then washed. Analytical procedures. The measurements of dry c e l l weight were made by a f i l t r a t i o n pore size f i l t e r s .
method using 0.45 Nm
In entrapped c e l l experiments, the
5-
_100
beads were dissolved in 100 ml of 0.2 M potassium phosphate buffer pH 7.5 ; 5 ml of the suspension were f i l t e r e d and the c e l l s t~vice washed with the buffer. The r e s p i r a t i o n rate of free or entrapped c e l l s , expressed as mg 02/g d.w.h, was measured with an oxygen electrode by f o l l o w i n g the oxygen consumption with a c h a r t - r e c o r d e r . The percentage of viable c e l l s was
0 0
r
I
100
200
Fig. 1 : Growth and v i a b i l i t y
w 300
of C a t h a r a n t h u s roseus
estimated by the erythrosine method described by
c e l l s in 2,4-D free medium a f t e r successive
Philipps and Henshaw (1977). Residual sugar, expressed
subcultures from normal medium. Closed symbols:
as glucose equivalen~was determined by an HPLC method
second t r a n s f e r ( f i r s t
using a NH2 s i l i c a column and a c e t o n i t r i l e - w a t e r (80/
analogous) ; open symbols : t h i r d t r a n s f e r .
20) as e l u t i o n solvent. Extraction of the alkaloids and q u a n t i t a t i v e determination of tryptamine, a j m a l i cine and serpentine were performed by the procedures previously described (Vinas and P a r e i l l e u x , 1982).
transfer results were
t r a t i o n (45 nM instead of 4.5 ~M) was selected f o r future use. Growth l i m i t a t i o n by reducing the supply of an essential n u t r i e n t , i . e . phosphate requirement was also attempted. The e f f e c t of various phosphate concentrations on c e l l growth showed a l i n e a r increase of biomass formation
RESULTS AND D I S C U S S I O N
over a 0-400 ~M range of the i n i t i a l
phosphate at day
Growth l i m i t a t i o n of free cell suspensions : effects
4 of the culture and over a 0-700 ~M range at day 7.
on c e l l v i a b i l i t y
The complete uptake of phosphate from the medium
and a l k a l o i d y i e l d
In an attempt to l i m i t cell growth in order to enable successful entrapment in Ca-alginate beads, hormonal or n u t r i e n t supply were investigated. When free cell suspensions grown i n i t i a l l y
on the normal medium were
subcultured into medium devoid of 2,4-D, the growth
observed f o r a l l concentrations investigated implied i n t r a c e l l u l a r storage of phosphate. I t can be thus calculated that below a c r i t i c a l
phosphate concentra-
tion corresponding to 40 pmol/g d . w . growth l i m i t a t i o n took place. The product y i e l d s obtained at day 7 f o r
was not affected by the omission of 2,4-D during the
various phosphate concentrations are given in Table I I .
first
Reducing the phosphate concentration resulted in
two subcultures. However, a lowered growth was
observed during the t h i r d subculture, with a p r o p o r t i o nal increase of c e l l death (Fig. I ) . I t can be assumed that the cessation of growth and the loss of c e l l v i a b i l i t y occured when the i n t r a c e l l u l a r concentration of 2,4-D f e l l below a treshold value, as suggested by Leguay and Guern (1977). Increasing the osmolarity of the medium with 200 mM mannitol reduced the rate of c e l l d i v i s i o n ; consequently, the c e l l death was
enhanced ajmalicine levels but decreased tryptamine levels ; no s i g n i f i c a n t modification appeared f o r serpentine. Similar results were reported by Knobloch and coworkers (1981, 1983) concerning the influence of phosphate on the growth and the accumulation of secondary compounds. As the metabolic a c t i v i t y of the c e l l s Table I : Maximum levels of tryptamine, a j m a l i c i n e and
shown to be delayed but not suppressed (results not
serpentine in normal or 2,4-D free medium
shown). Enhanced a j m a l i c i n e and serpentine accumulation
cultured c e l l s .
but lower tryptamine content were observed within the c e l l s during the second subculture in the 2,4-D free
A l k a l o i d content (~g/g dw.)
medium, compared with normal medium grown c e l l s (Table I ) . A s i m i l a r increase of indole a l k a l o i d accumulation
Product
Normal medium
has been reported by others (Knobloch and B e r l i n , 1980; Roustan et a l . ,
1982 ; Vinas and P a r e i l l e u x , 198Z). To
prevent c e l l death and to draw some p r o f i t from the stimulation of secondary metabolism r e s u l t i n g from decreased i n t r a c e l l u l a r 2,4-D, a lower medium concen-
2,4-D free medium (2 nd subculture)
Tryptamine
540
Ajmalicine
20
345 72
Serpentine
11
120
304 Table I I : Levels of tryptamine, ajmalicine and serpentine at day 7 of culture for various
biomass
(g/I)
10.
phosphate concentrations. 8-
Phosphate (NM)
Alkaloid content (~gl~ dw.) Tryptamine
Ajmalicine
Serpentine
0
135.5
30.2
2.6
25
175.2
23.3
4.3
50
210.0
21.5
3.2
100
247.3
17
3.7
250
387.75
12.2
6.0
500
420
7.6
5.0
1100
543
2.1
3.5
6_ •
&
ation .15
4_
-10 2_ -5 time(h)
o
I 200
0
~ ~ - - 0 I 400
4
! 600
Fig. 2 : Time course of biomass formation and respiratory activity for free (open symbols) and entrapped
has to be maintained for extended periods in entrapped
(closed symbols) cells of
cell u t i l i z a t i o n , a 100 ~M phosphate concentration was
C a t l ~ n t h u s roseus
grown
in the limiting medium.
selected. Based on these findings a growth limiting and product formation stimulating mediumwas formulated for subsequent cell entrapment studies, in which 45 nM 2,4-D and 100 ~M phosphate concentrations were used.
'1111"7 t r y p t a m i n e • ~7 a j m a l i c i n e
Biosynthetic activity of free or entrapped cells
(~Jg/g a.w,)
The time-courses of cell growth and respiration rates for both free and Ca-alginate immobilized cells using the above mentionnedmedium are given in Fig. 2. Polymer entrapped cells showed lower rates of biomass formation and i n i t i a l oxygen uptake comparedwith free cells, possibly due to a diffusion barrier as previously suggested by Jones and Veliky (1981). A constant respiratory activity was observed between 200 and 600 h of culture for immobilized cells, whereas the respiration rate decreased and cell lysis increased in free cell culture. The kinetics of product accumulation within
400-
the cells is shown in Fig. 3. Whereas the entrapment slightly affected the tryptamine content a more prolonged period of ajmalicine accumulation and higher amounts of this product were found in the entrapped cell culture. I t can be observed that the serpentine level was not enhanced in the limiting mediumfor either free or immobilized suspensions. Thus the entrapment stabilized markedly the v i a b i l i t y of the
i
I
I
20 4 Q O s e r p e n t i n e / 0
cells and resulted in enhancedajmalicine accumulation
i
{~g/gd.w.) '
over long periods of time. In these experiments the alkaloid content was also measured in the medium ; for instance, the maximumajmalicine concentration reached 80 ~gll (10 wg/g dw.) in the immobilized cell culture ; the higher value found in the free cell culture (700
L V
0
40o
600
Fig. 3 : Kinetics of product accumulation within free (open symbols) and entrapped (closed symbols)
~g/l, i.e. 70 ~g/g dw.) was an indication of a cell
cells of
lysis. The occurence of alkaloids in the medium, albeit in low amounts, suggested the a b i l i t y of the cells to
medium.
excrete partly their products. This a b i l i t y , i f possible to be exploited, has important consequencesfor alkaloid production. Promising studies have shown that the problem
200
a a t l ~ n t h u s roseus
grown in the limiting
305 of vacuolar storage should be overcome and thus
Brodelius P., Nilsson K. (1983) Eur.J.Appl.Microbiol.
enhanced product removal obtained using cell permeabi-
Biotechnol. 17 : 275-280.
l i z a t i o n (Brodelius and Nilsson, 1983), modulation of
Galun E., Aviv D., Dantes A., Freeman A. (1983) Planta
accumulation r a t i o by c o n t r o l l i n g the external pH
Med. 49 : 9-13.
(Renaudin, 1981) semi continuous and continuous mode
Gamborg O.L., M i l l e r R.A., Ojima K. (1968) Exp.Cell Res.
of operation (Brodelius and Nilsson, 1983, Vinas and
50 : 151-158.
P a r e i l l e u x , 1984). Inherent advantages f o r immobilized
Jones A., Veliky I.A. (1981) E u r . J . A p p l . M i c r o b i o l . B i o -
b i o c a t a l y s t seems to be evident in the l a t e r case.
technol. 13 : 84-89. Jirku V., Mocek T., Vanek T., Krumphanzl V., Kubanek V.
In conclusion, the results presented in t h i s paper establish the conditions required f o r c e l l entrapment
(1981) B i o t e c h n o l . L e t t . 3 : 447-450.
with long term s t a b i l i t y of the Ca-alginate matrix,
Knobloch K.H., Berlin J. (1980) Z.Naturforsch. 35C :
maintenance of the v i a b i l i t y
and enhanced biosynthetic
capacity of the c e l l s . Using such entrapped roseus
Cat~rantk~s
c e l l s , studies on the production of indole
a l k a l o i d s in a continuous flow reactor are now in progress.
551-556. Knobloch K.H., Beutnagel G., Berlin J. (1981) Planta 153 : 582-585. Knobloch K.H., Berlin J. (1983) Plant Cell Tissue Organ Cult. 2 : 333-340. Leguay J . J . , Guern J. (1977) Plant Physiol. 60 : 265-270.
ACKNOWLEDGEMENTS This work has in part been supported by the French M.R.T. The technical assistance of Mr C. Hormi6re is acknowledged. We are grateful to Dr. N.D. Lindley for revising the English manuscript.
Lindsey K., Yeoman M.M. (1983) J.Exp.Bot. 34 : 1055-1065. Lindsey K., Yeoman M.M., Black G.M., Mavituna F. (1983) FEBS L e t t . 155 : 143-149. Pareilleux A., Vinas R. (1984) Appl.Microbiol.Biotechnel. 19 : 316-320. Philipps R., Henshaw G.G. (1977) J.Exp.Bot. 28 : 785794.
REFERENCES
Renaudin J.P. (1981) Plant Sc.Lett. 22 : 59-69. Roustan J.P., Ambid C,, F a l l o t J, (1982) Physiol.V~g.
Brodelius P., Deus B., Mosbach K., Zenk M.H. (1979)
20 : 523-532.
FEBS L e t t . 103 : 93-97.
Shuler M.L. (1981) Ann.N.Y.Acad.Sci. 369 : 65-69.
Brodelius P., Nilsson K. (1980) FEBS L e t t . 122 : 312-
Vinas R., Pareilleux A. (1982) Physiol,Wg. 20 : 219-
316.
225.
Plant Cell Reports (1986) 5:302-305
© Springer-Verlag1986
Alkaloid accumulation in Ca-alginate entrapped cells of Catharanthus roseus: Using a limiting growth medium Florence Majerus and Alain Pareilleux D6partement de G6nie Biochimique et Alimentaire, UA-CNRS-N°544, Institut National des Sciences Appliqu6es, Avenue de Rangueil, F- 31077 Toulouse C6dex, France Received May 22, 1986 / Revised version received June 20, 1986 - Communicated by A. M. Boudet
ABSTRACT
continued growth of cells. There is evidence that plant
The limitation of growth of Cathavanthue r o s e u s cells
secondary metabolites are often produced at higher
was investigated with a view to their entrapment in a
concentration in slow growing or non-growing cultures
Ca-alginate matrix. An examination of the effects of
(Lindsey and Yeoman, 1983). This is the case for
lowered 2,4-D and phosphate concentrations on cell
Oathavanthus r o s e u 8 cells in which alkaloid accumulatio~
v i a b i l i t y and indole alkaloid biosynthesis enabled a
is restricted to the post exponential growth phase
growth limiting and product formation stimulating
(Pareilleux and Vinas, 1982). Obviously, i f the biocata-
medium to be designed. Entrappedcells showed a
l y t i c capacity of the cells could be maintained in
retention of both respiratory activity and biosynthetic
growth limiting conditions, the h a l f - l i f e of immobilized
capacity over an extended period of time comparedwith
systems should be increased, and the production yield
free cells. Evidence is presented which suggests that
enhanced. In this study, we investigated the capability
immobilization in Ca-alginate beads acts to stabilize
of growth-limited, suspendedand immobilized cells of
cells, resulting in enhancedproduct accumulation.
Oatharanthus roseus
ABBREVIATIONS
2,4-D : 2,4-dichlorophenoxyacetic acid; d.w.: dry weight.
MATERIALS AND METHODS
Cell suspension cultures. Cell suspension cultures of Cathavanthus
INTRODUCTION
Plant cell cultures have great potential in biochemical production and biotransformation reactions. With a view
to synthesize indole alkaloids.
roseus
(L.) G Don were maintained and
subcultured every week in the medium described by Gamborg et al. (1968) supplementedwith 4.5 ~M 2,4-D and 0.28 ~M kinetin and the addition of 3 % sucrose as
to enlarge production increased productivity of useful
the carbone source (basal medium). The suspensions were
compounds may be obtained using a continuous mode of
cultivated in the dark at 27°C in 250 ml Erlenmeyer
operation working over long periods. There are a number
flasks with 100 ml mediumper flask. When otheK media
of advantagesassociated with the use of immobilized
were tested, the cells were harvested by f i l t r a t i o n ,
or sequestered cells ; for instance, dilution rates in
washed and resuspended in the appropriate medium. The
excess of the growth rate of the culture can be used. A variety of methods for plant cell immobilization
30 g/l in the basal medium. The composition of the
have now been reported including entrapment in a
limiting mediumwas the sameas the basal mediumexcept
2,4-D free medium contained 50 g/l sucrose instead of
polymeric matrix (Brodelius et a l . , 1979 ; Brodelius
the concentrations of 2,4-D, phosphate and CaCl2 which
and Nilsson, 1980 ; Galun et a l . , 1983), covalent
were changed (45 nM, 100 ~M and 5 mM r e s p e c t i v e l y ) . The
linkage to polyphenylene oxide (Jirku et a l . , 1981),
f r e e l y suspended c e l l cultures were started with an
adsorption to microporous polyurethane foam (Lindsey
inoculum of 0.1-0.2 g d.w. per f l a s k . In entrapped c e l l
et a l . , 1983), entrapment in hollow fiber reactors
experiments, Erlenmeyer flasks were inoculated with 10 g
(Shuler, 1981). Most commonly, c e l l s are entrapped in
of beads (0.2 g d.w. per f l a s k ) Immobilization of c e l l s . Immobilization in Ca-alginate
a gel matrix, usually calcium a l g i n a t e . However, one of the problems with plant c e l l s immobilized in Ca-
was performed under aseptic conditions by the follow.img
alginate is the i n s t a b i l i t y of the matrix, due to a
method : c e l l s of a 7 day-old suspension were f i l t e r e d
Offprint requests to: A. Pareilleux
303 through a 800 ~m metal net and retained on a 40 ~m net. 250 g wet weight of c e l l s were suspended in 300 ml of
biomass
(g/I)
(%)
viability
,= z~
00
3 % a l g i n a t e dissolved in the l i m i t i n g medium and the suspension was added dropwise to the medium containing
10.
50 mM CaCI2. The beads were l e f t in t h i s solution f o r I h with s t i r r i n g and then washed. Analytical procedures. The measurements of dry c e l l weight were made by a f i l t r a t i o n pore size f i l t e r s .
method using 0.45 Nm
In entrapped c e l l experiments, the
5-
_100
beads were dissolved in 100 ml of 0.2 M potassium phosphate buffer pH 7.5 ; 5 ml of the suspension were f i l t e r e d and the c e l l s t~vice washed with the buffer. The r e s p i r a t i o n rate of free or entrapped c e l l s , expressed as mg 02/g d.w.h, was measured with an oxygen electrode by f o l l o w i n g the oxygen consumption with a c h a r t - r e c o r d e r . The percentage of viable c e l l s was
0 0
r
I
100
200
Fig. 1 : Growth and v i a b i l i t y
w 300
of C a t h a r a n t h u s roseus
estimated by the erythrosine method described by
c e l l s in 2,4-D free medium a f t e r successive
Philipps and Henshaw (1977). Residual sugar, expressed
subcultures from normal medium. Closed symbols:
as glucose equivalen~was determined by an HPLC method
second t r a n s f e r ( f i r s t
using a NH2 s i l i c a column and a c e t o n i t r i l e - w a t e r (80/
analogous) ; open symbols : t h i r d t r a n s f e r .
20) as e l u t i o n solvent. Extraction of the alkaloids and q u a n t i t a t i v e determination of tryptamine, a j m a l i cine and serpentine were performed by the procedures previously described (Vinas and P a r e i l l e u x , 1982).
transfer results were
t r a t i o n (45 nM instead of 4.5 ~M) was selected f o r future use. Growth l i m i t a t i o n by reducing the supply of an essential n u t r i e n t , i . e . phosphate requirement was also attempted. The e f f e c t of various phosphate concentrations on c e l l growth showed a l i n e a r increase of biomass formation
RESULTS AND D I S C U S S I O N
over a 0-400 ~M range of the i n i t i a l
phosphate at day
Growth l i m i t a t i o n of free cell suspensions : effects
4 of the culture and over a 0-700 ~M range at day 7.
on c e l l v i a b i l i t y
The complete uptake of phosphate from the medium
and a l k a l o i d y i e l d
In an attempt to l i m i t cell growth in order to enable successful entrapment in Ca-alginate beads, hormonal or n u t r i e n t supply were investigated. When free cell suspensions grown i n i t i a l l y
on the normal medium were
subcultured into medium devoid of 2,4-D, the growth
observed f o r a l l concentrations investigated implied i n t r a c e l l u l a r storage of phosphate. I t can be thus calculated that below a c r i t i c a l
phosphate concentra-
tion corresponding to 40 pmol/g d . w . growth l i m i t a t i o n took place. The product y i e l d s obtained at day 7 f o r
was not affected by the omission of 2,4-D during the
various phosphate concentrations are given in Table I I .
first
Reducing the phosphate concentration resulted in
two subcultures. However, a lowered growth was
observed during the t h i r d subculture, with a p r o p o r t i o nal increase of c e l l death (Fig. I ) . I t can be assumed that the cessation of growth and the loss of c e l l v i a b i l i t y occured when the i n t r a c e l l u l a r concentration of 2,4-D f e l l below a treshold value, as suggested by Leguay and Guern (1977). Increasing the osmolarity of the medium with 200 mM mannitol reduced the rate of c e l l d i v i s i o n ; consequently, the c e l l death was
enhanced ajmalicine levels but decreased tryptamine levels ; no s i g n i f i c a n t modification appeared f o r serpentine. Similar results were reported by Knobloch and coworkers (1981, 1983) concerning the influence of phosphate on the growth and the accumulation of secondary compounds. As the metabolic a c t i v i t y of the c e l l s Table I : Maximum levels of tryptamine, a j m a l i c i n e and
shown to be delayed but not suppressed (results not
serpentine in normal or 2,4-D free medium
shown). Enhanced a j m a l i c i n e and serpentine accumulation
cultured c e l l s .
but lower tryptamine content were observed within the c e l l s during the second subculture in the 2,4-D free
A l k a l o i d content (~g/g dw.)
medium, compared with normal medium grown c e l l s (Table I ) . A s i m i l a r increase of indole a l k a l o i d accumulation
Product
Normal medium
has been reported by others (Knobloch and B e r l i n , 1980; Roustan et a l . ,
1982 ; Vinas and P a r e i l l e u x , 198Z). To
prevent c e l l death and to draw some p r o f i t from the stimulation of secondary metabolism r e s u l t i n g from decreased i n t r a c e l l u l a r 2,4-D, a lower medium concen-
2,4-D free medium (2 nd subculture)
Tryptamine
540
Ajmalicine
20
345 72
Serpentine
11
120
304 Table I I : Levels of tryptamine, ajmalicine and serpentine at day 7 of culture for various
biomass
(g/I)
10.
phosphate concentrations. 8-
Phosphate (NM)
Alkaloid content (~gl~ dw.) Tryptamine
Ajmalicine
Serpentine
0
135.5
30.2
2.6
25
175.2
23.3
4.3
50
210.0
21.5
3.2
100
247.3
17
3.7
250
387.75
12.2
6.0
500
420
7.6
5.0
1100
543
2.1
3.5
6_ •
&
ation .15
4_
-10 2_ -5 time(h)
o
I 200
0
~ ~ - - 0 I 400
4
! 600
Fig. 2 : Time course of biomass formation and respiratory activity for free (open symbols) and entrapped
has to be maintained for extended periods in entrapped
(closed symbols) cells of
cell u t i l i z a t i o n , a 100 ~M phosphate concentration was
C a t l ~ n t h u s roseus
grown
in the limiting medium.
selected. Based on these findings a growth limiting and product formation stimulating mediumwas formulated for subsequent cell entrapment studies, in which 45 nM 2,4-D and 100 ~M phosphate concentrations were used.
'1111"7 t r y p t a m i n e • ~7 a j m a l i c i n e
Biosynthetic activity of free or entrapped cells
(~Jg/g a.w,)
The time-courses of cell growth and respiration rates for both free and Ca-alginate immobilized cells using the above mentionnedmedium are given in Fig. 2. Polymer entrapped cells showed lower rates of biomass formation and i n i t i a l oxygen uptake comparedwith free cells, possibly due to a diffusion barrier as previously suggested by Jones and Veliky (1981). A constant respiratory activity was observed between 200 and 600 h of culture for immobilized cells, whereas the respiration rate decreased and cell lysis increased in free cell culture. The kinetics of product accumulation within
400-
the cells is shown in Fig. 3. Whereas the entrapment slightly affected the tryptamine content a more prolonged period of ajmalicine accumulation and higher amounts of this product were found in the entrapped cell culture. I t can be observed that the serpentine level was not enhanced in the limiting mediumfor either free or immobilized suspensions. Thus the entrapment stabilized markedly the v i a b i l i t y of the
i
I
I
20 4 Q O s e r p e n t i n e / 0
cells and resulted in enhancedajmalicine accumulation
i
{~g/gd.w.) '
over long periods of time. In these experiments the alkaloid content was also measured in the medium ; for instance, the maximumajmalicine concentration reached 80 ~gll (10 wg/g dw.) in the immobilized cell culture ; the higher value found in the free cell culture (700
L V
0
40o
600
Fig. 3 : Kinetics of product accumulation within free (open symbols) and entrapped (closed symbols)
~g/l, i.e. 70 ~g/g dw.) was an indication of a cell
cells of
lysis. The occurence of alkaloids in the medium, albeit in low amounts, suggested the a b i l i t y of the cells to
medium.
excrete partly their products. This a b i l i t y , i f possible to be exploited, has important consequencesfor alkaloid production. Promising studies have shown that the problem
200
a a t l ~ n t h u s roseus
grown in the limiting
305 of vacuolar storage should be overcome and thus
Brodelius P., Nilsson K. (1983) Eur.J.Appl.Microbiol.
enhanced product removal obtained using cell permeabi-
Biotechnol. 17 : 275-280.
l i z a t i o n (Brodelius and Nilsson, 1983), modulation of
Galun E., Aviv D., Dantes A., Freeman A. (1983) Planta
accumulation r a t i o by c o n t r o l l i n g the external pH
Med. 49 : 9-13.
(Renaudin, 1981) semi continuous and continuous mode
Gamborg O.L., M i l l e r R.A., Ojima K. (1968) Exp.Cell Res.
of operation (Brodelius and Nilsson, 1983, Vinas and
50 : 151-158.
P a r e i l l e u x , 1984). Inherent advantages f o r immobilized
Jones A., Veliky I.A. (1981) E u r . J . A p p l . M i c r o b i o l . B i o -
b i o c a t a l y s t seems to be evident in the l a t e r case.
technol. 13 : 84-89. Jirku V., Mocek T., Vanek T., Krumphanzl V., Kubanek V.
In conclusion, the results presented in t h i s paper establish the conditions required f o r c e l l entrapment
(1981) B i o t e c h n o l . L e t t . 3 : 447-450.
with long term s t a b i l i t y of the Ca-alginate matrix,
Knobloch K.H., Berlin J. (1980) Z.Naturforsch. 35C :
maintenance of the v i a b i l i t y
and enhanced biosynthetic
capacity of the c e l l s . Using such entrapped roseus
Cat~rantk~s
c e l l s , studies on the production of indole
a l k a l o i d s in a continuous flow reactor are now in progress.
551-556. Knobloch K.H., Beutnagel G., Berlin J. (1981) Planta 153 : 582-585. Knobloch K.H., Berlin J. (1983) Plant Cell Tissue Organ Cult. 2 : 333-340. Leguay J . J . , Guern J. (1977) Plant Physiol. 60 : 265-270.
ACKNOWLEDGEMENTS This work has in part been supported by the French M.R.T. The technical assistance of Mr C. Hormi6re is acknowledged. We are grateful to Dr. N.D. Lindley for revising the English manuscript.
Lindsey K., Yeoman M.M. (1983) J.Exp.Bot. 34 : 1055-1065. Lindsey K., Yeoman M.M., Black G.M., Mavituna F. (1983) FEBS L e t t . 155 : 143-149. Pareilleux A., Vinas R. (1984) Appl.Microbiol.Biotechnel. 19 : 316-320. Philipps R., Henshaw G.G. (1977) J.Exp.Bot. 28 : 785794.
REFERENCES
Renaudin J.P. (1981) Plant Sc.Lett. 22 : 59-69. Roustan J.P., Ambid C,, F a l l o t J, (1982) Physiol.V~g.
Brodelius P., Deus B., Mosbach K., Zenk M.H. (1979)
20 : 523-532.
FEBS L e t t . 103 : 93-97.
Shuler M.L. (1981) Ann.N.Y.Acad.Sci. 369 : 65-69.
Brodelius P., Nilsson K. (1980) FEBS L e t t . 122 : 312-
Vinas R., Pareilleux A. (1982) Physiol,Wg. 20 : 219-
316.
225.
