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Plant Cell Reports (1985) 4: 220- 223

PlantCell
Reports

Springer-Verlag 1985

Cultivation of cell cultures of Berberis wiisonae in 20-1 airlift bioreactors


M. Breuling, A. W. Alfermann, and E. Reinhard
Pharmazeutisches Institut (Pharmazeutische Biologic), Universit~t Ttibingen, Auf der Morgenstelle 8, D-7400 Ttibingen, FRG
Received June 3, 1985 / Revised version received July 11, 1985 - Communicated by W.Barz

ABSTRACT
S u s p e n s i o n c u l t u r e s of B e r b e r i s w i l s o n a e
produce 4 berberine-type alkaloids: berberine, p a l m a t i n e , c o l u m b a m i n e
and J a t r o r rhizine. In particular the f o r m a t i o n of the
phenolic alkaloids c o l u m b a m i n e and Jatrorrhizine and of berberine proves to be dependent on the concentration of dissolved oxygen. W i t h h i g h e r a e r a t i o n rates, b e r b e r i n e
and J a t r o r r h i z i n e y i e l d s can be i n c r e a s e d
c o n s i d e r a b l y . T h u s we r e a c h e d an a l k a l o i d
y i e l d of m o r e t h a n 3 g x l- w i t h 50% d i s solved oxygen tension in the medium. As far
as we k n o w this is one of the b e s t r e s u l t s
in f e r m e n t i n g of a l k a l o i d - p r o d u c i n g
cell
cultures.
ABBREVIATIONS
P02, d i s s o l v e d o x y g e n c o n c e n t r a t i o n in %
s a t u r a t i o n ( u s i n g air); HPLC, h i g h - p e r f o r mance liquid chromatography; vvm, v o l u m e air
x v o l u m e m e d i u m -I x m i n u t e - l ; rpm, r e v o l u tions per minute; IAA, indole-3-acetic acid;
2,4-D, 2,4-dichlorophenoxy acetic acid.

Berberine is the m a j o r alkaloid in the root


b a r k of the plant; in cell c u l t u r e s , h o w ever, j a t r o r r h i z i n e p r e d o m i n a t e s (Hinz et
al. 1981, R o t h e n b e r g e r 1982).
In 1981 H i n z et al. r e p o r t e d a y i e l d of
a b o u t 1.7 g a l k a l o i d s x 1-1 m e d i u m
from
Berberis stolonifera using 100-ml flasks. By
s e l e c t i n g a h i g h l y p r o d u c t i v e cell line of
Berberis wilsonae in 1982 Rothenberger was
a b l e to i n c r e a s e the a l k a l o i d y i e l d up to
2.26 g x 1-1 m e d i u m . E x p e r i m e n t s w i t h 1-1
f l a s k s r e s u l t e d in a d e c r e a s e in the a l k a loid c o n t e n t of a b o u t 25% as c o m p a r e d w i t h
the results in 100-ml flasks.
A l k a l o i d f o r m a t i o n as a f u n c t i o n of d i s solved oxygen tension has been discussed for
C o p t i s ~ a p o n i c a by Y a m a d a et al. (198!) and
by S a t o et al. (1982).
W i t h i n the scope of our work about a l k a l o i d
f o r m a t i o n in f e r m e n t i n g cultures of Berberis
w i l s o n a e , the p r e s e n t s t u d y w i l l r e p o r t a
f e w a s p e c t s of the i n f l u e n c e of d i s s o l v e d
oxygen tension in 20-1 airlift reactors.

MATERIALS AND METHODS

INTRODUCTION

Culture conditions

Protoberberine-type
i s o q u i n o l i n e alkaloids
are c h a r a c t e r i s t i c
of the B e r b e r i d a c e a e .
M e m b e r s of this family are used in medicine,
e s p e c i a l l y in the F a r - E a s t , b e c a u s e of the
anti-bacterial and a n t i - i n f l a m m a t o r y properties of t h e i r a l k a l o i d s (Kondo 1976). In
addition, berberine-type alkaloids are k n o w n
to be c a p a b l e of i n d u c i n g c y t o s t a t i c a c t i v i t y in m a c r o p h a g e s in v i t r o ( K u m a z a w a et
al. 1984).
Berberis
s p e c i e s s y n t h e s i z e 4 m a i n berberine-type a l k a l o i d s : b e r b e r i n e , c o l u m b a m i n e , j a t r o r r h i z i n e and p a l m a t i n e (fig. I,
I k r a m 1975).

Cell cultures of Berberis wilsonae Hemsl. &


Wils., line WSG3, w e r e u s e d for t h e s e investigations. This high alkaloid producing
cell line was selected
by R o t h e n b e r g e r
(1982) f r o m B e r b e r i s w i l s o n a e c a l l u s cultures because of its dark orange colour. The
c e l l s w e r e s u b c u l t i v a t e d e v e r y I0 d a y s by
i n o c u l a t i n g 36 g c e l l s (fresh w e i g h t ) into
300 ml of m e d i u m in i-i E r l e n m e y e r - f l a s k s .
T h e y w e r e k e p t on r o t a r y s h a k e r s (120 r p m )
in the d a r k at 24Oc. The b a s a l m e d i u m of
M u r a s h i g e and Skoog (1962) was used w i t h the
f o l l o w i n g modifications: 40 g. x i -I sucrose,
0.2 ~ g x i -i IAA, 0.2 m g x 1 -I 2,4-D, 2.0 mg
x i- kinetin.
Eight 10-day-old flasks were used to inoculate the W a h l
(1977) l a b o r a t o r y a i r l i f t
r e a c t o r c o n t a i n i n g the s a m e m e d i u m u s e d
d u r i n g p r e c u l t u r e w i t h the s u c r o s e c o n c e n t r a t i o n r e d u c e d to 20 g x I -I. The w o r k i n g
v o l u m e w a s k e p t c o n s t a n t at 19 1 by a d d i n g
s t e r i l e w a t e r . If n e c e s s a r y an a n t i f o a m
m a t e r i a l w a s a d d e d (2.5 g p o l y p r o p y l e n g l y -

R10~

R1.R2=GH2 berberine

R20.,.,I.~ ~.1

~:CH3,R~CH
3 polmatine

~~I#=CH3,R'H

columbomine

~=H ,R~CH3 jQtro~hizine

F i g u r e I. The 4 m a i n a l k a l o i d s
wilsonae.

Offprint requests to." M. Breuling

of B e r b e r i s

221
col, m o l w e i g h t 2025, E. M e r c k , D a r m s t a d t ,
FRG, x I - water). B e c a u s e of the g r a d u a l
d e p l e t i o n of the c a r b o n s o u r c e , a 30% g l u cose solution (w/v) was fed to the reaction
mixture
f r o m day 6 on. F e r m e n t a t i o n
was
carried out w i t h a constant aeration rate or
a constant dissolved oxygen concentration
(P02). The pO 2 w a s m e a s u r e d in s i t u w i t h a
p o l a r o g r a p h i c o x y g e n e l e c t r o d e (Dr. W. Ingold, U r d o r f , S w i t z e r l a n d ) and r e g u l a t e d
u s i n g an o x y g e n c o n t r o l u n i t f r o m B. B r a u n
(Melsungen, FRG).
A n a l y t i c a l procedures
i00 mg of the dried and p o w d e r e d tissue were
extracted under reflux with i0 ml methanol.
A f t e r c o o l i n g the e x t r a c t was f i l t e r e d and
diluted with m e t h a n o l 1:20.
The a l k a l o i d c o n t e n t of the s o l u t i o n was
a n a l y z e d by H P L C ( B e c k m a n G r a d i e n t L i q u i d
Chromatograph
M o d e l 332) u s i n g a 250 x 4.6
mm stainless steel column with Nucleosil
5 C 1 8 ( M a c h e r e y & Nagel, D ~ r e n , FRG) as the
stationary phase. The liquid phase was water
a d j u s t e d to pH3 w i t h H 3 P O 4 and 84% a c e t o nitrile redistilled in w a t e r adjusted to pH B
w i t h H 3 P O 4. We u s e d a m o d i f i c a t i o n of RHf ~
fer,s gradient (personal c o m m u n i c a t i o n 1984)
as s h o w n in fig. 2.
The flow rate was 1.5 ml x min. -I, m e a s u r i n g
w a v e length 228 rim, injection volume 20 ul.

0 % B (Acetonitrite,84%, pH 3)
96-

4431+"

27"
6 ......

' ....

1'0 ....

' -

2'0"

" "" ' " " " 3'0

" " 'train]

T h i s s i m p l e m e t h o d r e s u l t s in g o o d s e p a r a t i o n and q u a n t i f i c a t i o n of the f o u r a l k a loids.


RESULTS AND D I S C U S S I O N
It has b e e n s h o w n by v a r i o u s a u t h o r s t h a t
airlift reactors are suitable for secondary
p r o d u c t f o r m a t i o n by p l a n t c e l l c u l t u r e s
(see i.e. W a g n e r and V o g e l m a n n 1977, F o w l e r
1981, S m a r t and F o w l e r 1984). O n l y at h i g h
cell d e n s i t i e s can m i x i n g p r o b l e m s o c c u r
t h a t m i g h t m a k e it a d v a n t a g e o u s to u s e a
s t i r r e d - t a n k r e a c t o r ( T a n a k a 1981, U l b r i c h
et al. 1985, S p i e l e r 1985).
In p r e l i m i n a r y e x p e r i m e n t s
we f o u n d t h a t
airlift reactors can be used for cultivating
Berberis cell cultures for alkaloid production. H e r e we w i l l d e m o n s t r a t e the i m p o r t a n c e of c o n t r o l l i n g the d i s s o l v e d o x y g e n
t e n s i o n (P02) in the b i o r e a c t o r in o r d e r to
achieve optimal product yields.
Influence

of pO 2 on cell g r o w t h

T a b l e i s h o w s that a d i s s o l v e d o x y g e n tension b e t w e e n 20 and 50% saturation using air


for aeration
h a s no i n f l u e n c e
on c e l l
growth~ The cell yield on day 14 is about 25
g x 1 -i, the s p e c i f i c g r o w t h rate
during
the l o g a r i t h m i c p h a s e of g r o w t h is b e t w e e n
0.13 and 0.15 days -i , and the d o u b l i n g t i m e
is b e t w e e n 4.6 and 5.5 days. By feeding more
g l u c o s e to the r e a c t i o n m i x t u r e d u r i n g a
cultivation time of 20 days, dry weights of
m o r e t h a n 40 g x i -I can be a c h i e v e d (see
a l s o figs. 4 and 5).
U n d e r s u c h c o n d i t i o n s d i f f e r e n c e s in c e l l
yields can be observed as a result of insufficient m i x i n g with low aeration rates (only
up to 0.3 v v m or a pO 2 of 20%). T h e h i g h e r
aeration rates necessary to guarantee 40 or
50% pO 2 a l s o r e s u l t in a g o o d m i x i n g of the
culture
b r o t h . 0.5 v v m a n d m o r e c a u s e s
g r o w t h reduction because of shear stress and
high ventilation (cf. Smart
and F o w l e r 1 9 8 4 ~

aeration rate
dissolved oxygen
spec growth rate
doubling time
dry weight at day1&

(vvm)
0.1-0.3 0.1-0./+ 0+1-0.5
{%p02)
20
40
50
(days-1)
Q15
0.15
0.13
(days)
4.6
4.6
5.5
(gxl-l)
25.5
25.1
24.8

T a b l e I. G r o w t h d a t a at v a r i o u s
oxygen concentrations.

dissolved

Effect of pO 2 on alkaloid f o r m a t i o n

r""

"

....

' ....

....

'tm+n]

columbamine
J: J a t r o r r h i z l n e
p: p a l m a t i n e
b: berberine
F i g u r e 2.a: P e r c e n t a g e of a c e t o n i t r i l e in
the l i q u i d phase. R i n s i n g
cycle between
m i n u t e 25 and 35. b: H P L C - c h r o m a t o g r a m
of
the 4 p r o t o b e r b e r i n e alkaloids.
c:

Fig. 3 shows that increasing the oxygen tension and a e r a t i o n rate enhances the a c c u m u lation of berberine, c o l u m b a m i n e and jatrorrhizine, w h e r e a s p a l m a t i n e is only affected
to a v e r y m i n o r e x t e n t . T h i s r e f l e c t s the
close b i o s y n t h e t i c i n t e r r e l a t i o n s h i p of the
f i r s t t h r e e a l k a l o i d s ; the f i n a l s t e p s in
p a l m a t i n e b i o s y n t h e s i s may be s o m e w h a t divergent. W h e r e a s fig. 3 only shows the m a x i mal a m o u n t of alkaloids, fig. 5 d e m o n s t r a t e s
the k i n e t i c s of a c c u m u l a t i o n
for the f o u r
a l k a l o i d s d u r i n g the g r o w t h c y c l e of the
cells. M a x i m a l berberine a c c u m u l a t i o n occurs
v e r y e a r l y (days 2-4) d u r i n g the lag p h a s e
in the g r o w t h cycle, d i m i n i s h i n g
w h e n columbamine
and j a t r o r r h i z l n e a c c u m u l a t i o n
i n c r e a s e d u r i n g the l o g a r i t h m i c and e a r l y

222

mg g-1

mg g.-1

a, O~ P2 :
1 (3 20%
~81

132=
40'/,,/~

132:
50%

I 1I

s t a t i o n a r y g r o w t h phase. This, too, d e m o n strates the intermediate function of berberine in columbamine and Jatrorrhizine biosynthesis, as was found in Beecher and Kelleher,s enzymatic studies (1983).
The maximum
Jatrorrhizine
accumulation
(10.1%) in percent of dry m a t t e r can be
o b s e r v e d on day 15. A l k a l o i d b i o s y n t h e s i s ,
however, continues until day 20, as is shown
in fig. 4.

ao

20t ~r~
-5~I0

o~

bpc j

bpc j

vvm

wm=

00~

vvm= ,,1

bpc j

bpc j

Figure 3. The dependence


tion on various aeration
a: increasing pO 2.
b: increasing vvm.

bpc j
I\

mg g-1
80

ill

"

60~
N
l..

~o ~,

b)c j

of alkaloid
rates.

forma-

20

g1-1

gl-1
4

&O

I
0

3I

30
-

C@

E
2~
o

20
L.

x
0

o
0

10

12

16 20

deys

Figure 4. The yields in dry weight and total


a l k a l o i d s show parallel courses during the
whole fermentation cycle.

12

16 20

doys

Figure 5. The kinetics of alkaloid accumulation d u r i n g the g r o w t h cycle and the yield
in dry w e i g h t at P02=50% in the culture
broth.
Because of the significant increase in cell
dry w e i g h t there is still an i n c r e a s e in the
to{al a l k a l o i d yield up to m o r e than 3 g x
I- . To our knowledge, these are the highest
alkaloid yields from plant cell cultures in
bioreactors.
Although the oxygen demand of plant cells is
quite low when compared with microorganisms
the e x p e r i m e n t s p r e s e n t e d here show very
clearly that it may be very i m p o r t a n t to
control the oxygen tension in the bioreactor
in order to obtain optimal secondary product
yields f r o m plant cell cultures. S i m i l a r
results were o b t a i n e d by S p i e l e r et ai.
(1985). M o r e o v e r , it can be seen that by
p r o v i d i n g the a p p r o p r i a t e c u l t u r a l conditions it is possible to improve the product
yields in bioreactors substantially as compared with the results in shake flasks.
ACKNOWLEDGEMENTS
We w o u l d like to thank Dr. M. R G f f e r (Munich, FRG) for her kind advice and v a l u a b l e
information on alkaloid separation by HPLC.

223
This w o r k was s u p p o r t e d by the B u n d e s m i n i sterium fGr Forschung und Technologie.

Sato F, Endo T, Mashimoto T, Yamada Y (1982)


in: F u J i w a r a E (ed) Plant Tissue C u l t u r e
Tokyo, p 319

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531

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