Zbl. Bakt. II. Abt., Bd. 133, S.

551-556 (1978)

[National Research Centre, Dokki, Cairo, Egypt, and Institute of Microbiology, Czechoslovak Aca-
demy of Sciences, Prague.]

Variability of Requirements to Fermentation Media III Claviceps

purpurea Strains

O. HAMED EL-SAYED and Z. REHACEK

With 3 Figures

Summary
The alkaloid-synthesizing activity of the saprophytic Claviceps purpurea strains was Yariable.
This fact was partly due to different requirements of the cultures to nutriticnal conditions. Not only
single strains, but also formation of intra- and extracellular alkaloids differed in this respect. In case
of extracellular alkaloids, the influence of medium was more expressive. A positive correlation be-
tween accumulation of cell polysaccharides and the level of intracellular alkaloids was evident. Con-
stant or decreasing cell proteins were favourable for alkaloid formation.

Zusammenfassung
In saprophytischen Claviceps purpurea-Stammen wurde eine variable Aktivitat der Alkaloid-
bildung festgestellt. An dieser Variabilitat widerspiegeln sich die Unterschiede in den Al~spriichen
der einzelnen Stamme an das Medium. Auch die Biosynthese der intra- und extrazellularen Alkaloide
war von der Zusammensetzung des Nahrmediums abhangig. 1m Faile der extrazellularen Alkaloide
war der Einflu13 des Mediums besonders deutlich. - Es wurde eine positive Korrelation zwischen der
Speicherung von Zellpolysacchariden und intrazellularen Alkaloiden beobachtet. - Ein gleichblei-
bender oder sinkender Zellproteinspiegel war fUr die Alkaloidbildul'g giinstig.

The first convincing demonstration of a production of ergot alkaloids in vitro

(ABE 1951) was followed by a series of papers on the saprophytic nutritional require-
ments. However, it is still impossible to make any generalization of the factors,
influencing alkaloid formation, because of great differences among the strains. In
addition, nutritional conditions that favour maximum primary shunt metabolism in
a given case may not be expected to favour maximum alkaloid synthef'is (REHACEK
1971). In this investigation, submerged cultures of three strains of Claviceps purpurea
(Fr.) Tu!. on different fermentation media were examined. The purpose of this presen-
tation was to gain information that may lead to better understanding of the physio-
logy of alkaloid biosynthesis.

Materials and Methods

Organisms and culture conditions
The following saprophytic strains of Claviceps purpurea (Fr.) Tul. were used in this work: Pia 4,
Plo 2, and ATCC 14934. The strain Pia 4 (HRONES and KYBAL 1962) was prepared from the ground
plectenchymatous tissues of the sclerotia (lhclcovA and REH..\.CEK 1968).

36"
552 O. H. EL-SAYED and Z. REHACEK

Nutrient media of the following types were used in fermentation experiments:

A - mannite, ammonium succinate (Bu'LooK and BARR 1968);
B - mannite, glucose, ammonium succinate (VINING 1970);
C - mannite, sucrose, ammonium succinate, yeast extract (KAPLAN et al. 1969);
D - sorbitol, ammonium succinate (O'BAYASHI and KANIE 1966);
E - sucrose, ammonium citrate (MAIER et al. 1971);
F - glucose, ammonium phosphate (KOBEL et al. 1964).

The fermentation experiments were carried out in 300 ml Erlenmeyer flasks, containind 100 ml
of medium. The flasks were incubated at 24° ± 1 °C on a rotary shaker, operating at 240 rpm with
a 5.5 cm stroke. Each flask was shaken and inoculated with washed and blended (15 sec) vegetative
cell inoculum, equivalent to 10 mg of dry weight.
The inoculum was obtained from 5-day-ok1 shaken cultures, grown in 4.5 % of malt extract broth
(Difco) in distilled water. The experimental design consisted of growing the cultures in a rich medium
that did not favour alkaloid production and of subsequent transfer to a nutritionally poor medium
in which alkaloids are known to be formed.

Analytical methods
Protein was determined by the method of LOWRY et al. (1951), total alkaloids according to ROEDER
et al. (1967). The alkaloid results were expressed as ergometrine equivalents. For determination of
total polysaccharides by the phenol sulphuric acid method (Du BOIS et al. 1956), the mycelia were
washed with distilled water, then with ethyl alcohol (80%), and homogenized for 5 minutes (high
speed rate). The mycelial pellet was resuspended in 10 ml of distilled water and homogenized again
for 5 minutes. The final suspension was taken for determination of total polysaccharides.

Results and Discussion

Three investigated strains of C. purpurea produced a mixture of extra- and intra-
cellular alkaloids. This alkaloid mixture comprised partly clavines and simple lysergic
acid derivatives (extracellular), partly peptide alkaloids (intracellular). The amount
of intracellular alkaloids was lower than that of extracellular ones (Table 1).
The alkaloid-synthesizing activity of the strains was variable. This fact was due
to different susceptibility of producing cells to the composition of fermentation me-
dium. Not only single strains (Fig. 1a, b, c; 2) but also formation of intra- and extra-
cellular alkaloids (Table 1) differed in their claims to nutritional conditions. The in-
fluence of medium was more expressive in case of extracellular alkaloids than in case
of intracellular ones.
Strain Plo-2, with the highest alkaloid-synthesizing activity (Table 1), was observed
to be most medium-dependent, especially on its sugar component. In this strain man-
nite (medium A) was a very suitable carbon source during synthesis of extracellular
alkaloids. The positive role of mannite was reduced when also glucose or sucrose was
present (medium B, C). In contrast to strain Plo-2, strain Pla·4 exerted intensive
alkaloid synthesis both in medium containing mannite together with glucose (B) and
in medium with sucrose (E). Strain ATCC 14934 was very active on medium with
glucose (F) and on medium with sucrose (E).
Differences in susceptibility to medium were also noted in extra· and intracellular
alkaloid synthesis (Table 1). In strain Plo-2, medium with mannite (A) increased the
formation of extracellular alkaloids, while medium with sorbitol (D) and that with
glucose (F) were suitable for formation of intracellular alkaloids. On the other hand,
in strain ATCC 14934 extracellular alkaloid formation was intensified in medium with
glucose (F), while medium with mannite and glucose (B) gave rise to synthesis of
intracellular alkaloids.
 Variability of Requirements to Fermentation Media 553

Table 1. Some characteristics of mycelial) of Glaviceps purpurea strains in different fermentation

media

Strain ~Iedium Dry weight Proteins Polysaccharides Alkaloids (pg . 10 mg D.W. -1)
mg' 10 ml- l pg . mg pg . mg D.W.-l
D.W.-l Intracellular Extracellular Total

Pla-4 A 12 41 24 3 80 83
B 2 28 25 2 400 402
C 5 34 20 2 210 212
D 3 7 24 2 240 242
E 4 55 22 2 380 382
F 2 20 20 2 300 302

Plo-2 A 1 9 15 5 900 905

B 7 17 16 5 130 135
C 6 21 21 3 170 173
D 1 13 36 60 600 660
E 2 6 6 8 430 438
F 1 6 :37 60 600 660

ATCC A II 82 22 5 80 85
14934 B 44 25 30 50 120 170
C 16 17 20 5 80 85
D 22 7 33 3 220 223
E 22 28 55 20 400 420
F 12 19 22 10 540 550

I) 9-day-old shaken cultures with maximum alkaloid yield.

The physiological conditions necessary for the culture growth differed from those
that determined the start of alkaloid synthesis. Increasing alkaloid formation was
negatively correlated with the growth-rate capacity. Media inducing intensive culture
growth were usually unsuited to alkaloid formation (Table 1).
During alkaloid-producing phase different cell components were formed with differ-
ent intensities. Increasing cell-protein level corresponded to relative decrease of the
alkaloid-synthesizing activity. Constant or decreased cell proteins were favourable
for alkaloid formation and indicated a non-proliferating culture (Fig. 3). In the light
of this fact, and according to previous data (Bu'LoCK and BARR 1968, KAPLAN et al.
1969, thHACEK et al. 1971) which demonstrated the occurrence of no protein
synthesis at a constantly decreasing rate and the almost invariable level of cell pro-
teins as well as the low protease activities, we suggest a positive association between
the rate of protein turnover and alkaloid-synthesizing activity.
The level of cell polysaccharides was less variable than that of cell proteins. It is
noteworthy that a positive correlation between accumulation of both cell polysaccha-
rides and intracellular alkaloids was evident.
The results of this presentation demonstrate partly a variability of O. purpurea
in its claims to fermentation medium, partly some consequences of this property in
physiology of the culture and alkaloid formation. Our data are in harmony with the
hypothesis (TABER and VINING 1963, TABER 1964, Bu'LoCK 1965) that nutritional
conditions which favour maximum primary shunt metabolism in a given case are
not expected to favour maximum secondary shunt metabolism.
 554 o. H. EL-SAYED and Z. REHACEK

150

140
PLO-2

~ 100 -7'100
-;
E E
Q Q
.;. Do
~
~

til til
Q Q

<5 (5
oJ oJ
cl: cl:
:ll: 50 :ll: 50
...J ...J
cl: cl:

0 0

0 3 5 6 7 9 0 3 5 6 7 9
TINE (days) TIME (days)
Ia Ib

ISO
ATCC 14934

T
eQ
.;.
100
3 100
til
Q

9
cl:
:ll:
...J
cl:

0:
eQ
cl: .;.
:3 50
...J
350
til
Q
~
0 (5
cl: ...J
0: cl: PLO-2
I- :ll:
x ...J
&IJ cl:

0 0

0 3 5 6 7 9 0 3 5 6 7 9
TIME (days) TIME (days)
1c 2
 Variability of Requirements to Fermentation Media 555

=-

~~I
STRAIN ~~,
Z
~ filol
0 4Q ATCC 14934 PLA-4 PLO-2 15~:
l&J lll:OO a:: '0
~ oJ:;
4- a.j.

E
150
100 ---....... / ,, ,,- , ----............. -
100

SO
....,---",,. ...,-_ ....... / ......
0 0

ISO 100

C
100
A ,
I
~ ,
50
...
.... _-""
I
... .,-
........ _--, ::.::----><
0 0

ISO 100

c
100

0
L ~
... _---_ ....
,,
-----./
------ ... -....
50

0
150 100

100
B
===-<:'""... -- )-/ .... --' ~
---------
50

0 0

150 100

.----
100
0 50
~ ... ....
0
--------, " ... ----- ...... ......... _---- 0
150 100
100
F ,, 50
~
.... ...
~
~
----" .... _--"'"
0 0
o 5 7 9 5 7 9 5 7 9
TIME (days)

Fig. 3. The amount of cell proteins and total alkaloids of C. purpurea strains, grown in different fer·
mentation media.
AL - alkaloids
P - cell proteins

Fig. I a, b, c. Total alkaloids, produced by C. purpurea strains in different fermentation media.

I a·strain Pla·4, I b·strain PI6·2, I c·strain ATCC 14934. A, B, C, D, E, F - fermentation media as
indicated in Materials and Methods.

Fig. 2. Alkaloid yields of C. purpurea strains on mannite ammonium·succinate medium (A).

556 O. H. EL-SAYED and Z. REHACEK, Variability of Requirements to Fermentation Media

References
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1965, p. 61-71.
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Authors' addresses:
Dr. O. HAMED EL-SAYED, National Research Centre, Dokki, Cairo, Egypt, and RNDr. Z. REHACEK,
DSc., Institute ofMicrobiology, Czechoslovak Academy of Sciences, Budeovicka 1083, 14220 - Praha 4-
Krc(CSSR).

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