Appl Microbiol Biotechnol (1987) 27 : 117-- 120
AFtcrobiology
Correlation between growth and ergot alkaloid biosynthesis
in Claviceps purpurea batch fermentation
S. Militia, M. Kremser, V. Gaberc-Porekar, M. Didek-Brumec, and H. So~i~
Boris Kidri~ Institute of Chemistry and LEK, Pharmaceutical and Chemical Works, Ljubljana, Yugoslavia
Summary. Following the growth kinetics of a C.
purpurea ergotoxine-producing strain it was ob-
served that alkaloid synthesis and alkaloid distri-
bution depend on fungal growth velocity during
idiophase. Formation of extracellular alkaloids is
favoured by higher fungal growth velocity, while
intracellular alkaloids begin to accumulate at a
lower rate of growth. Simple lysergic acid deriva-
tives prevail among extracellular alkaloids,
whereas ergotoxines predominate among intracel-
lular alkaloids. By varying cultivation conditions,
by feeding the culture, or by varying the inoculum
size, alkaloid composition can be influenced
within the limits of strain capabilities.
Introduction
Ergot alkaloids produced during submerged culti-
vation of Claviceps purpurea are usually presented
as the sum of intra- and extracellular alkaloids.
However, peptide alkaloids tend to be located in
a higher proportion intracellularly within the my-
celium (Amici et al. 1967), while simple lysergic
acid derivatives (LAD) are released into the me-
dium (Arcamone et al. 1961). Authors explained
this phenomenon as the consequence of lower wa-
ter solubility of peptide ergot alkaloids in com-
parison to LAD. Hamed and Reha6ek (1978)
found, however, that formation of intra- and ex-
tracellular alkaloids is partly due to the different
nutritional requirements of C. purpurea.
Investigating some factors which could in-
fluence the fungal growth kinetics (inoculum size,
Offprint requests to: H. So6i6, Boris Kidri6 Institute of Chem-
istry, 61 115 Ljubljana, P.O. Box 30, Hajdrihova 19, Yugo-
slavia
Applied
Biotechnology
© Springer-Verlag 1987
feeding the culture), we observed that changes in
fungal growth velocity (dX/dt) may involve
changes in alkaloid qualitative composition, as
well as their intra- and extracellular distribution.
Materials and methods
Microorganism. Claviceps purpurea conidial strain, designated
L-17, was isolated from a selected parasitic strain. It is capable
of producing ergotoxine alkaloids in saprophytic cultures.
Cultivation procedure. The organism was maintained on su-
crose-peptone agar plates. Liquid seed medium was inocu-
lated with 21-day-old homogenized colonies, about 15 mg of
biomass dry weight was used to inoculate 50 ml of seed me-
dium in a 250-ml Erlenmeyer flask. After 4 days of cultivation
at 24°C on a rotary shaker at 240 rpm, 700 ml of inoculum
containing 15 g/1 of dry wt. was used for inoculating a 7-1 la-
boratory fermentor (Chemap). The producing stage proceeded
under the following operating conditions: 600 rpm, 3 l/rain of
air flow, at 24°C, for 14 days.
Culture media. The seed and the production media were pre-
pared according to French pat. (1976).
Seed media contained (g/l): sucrose 100, ammonium ox-
alate 3, proflo 10, KH2PO4 0.25, MgSOa-7H20, 0~25,
ZnSO4-7 H20 0.0068, FeSO4-7 H20 0.0166, distilled water to
1 1. The pH of the medium was 6.2.
The production medium was composed of (g/l): sucrose
240, ammonium oxalate 9.6, urea 1.7, KH2PO4 0.625,
MgSO4"7 H20 0.625, ZnSO4-7 H20 0.010, FeSO4.7H20 0.025
and distilled water to 1 1. The pH was 6.2.
Biomass determination. Culture broth (50ml) was filtered
through weighed filter paper on a Biichner funnel, the myce-
lium was washed twice with distilled water and dried at 85 °C
to constant weight.
Alkaloids. These were determined in fermentation broth fil-
trate and in mycelium separately, by van Urk reagent (Agurell
1966). Intracellular alkaloids were previously extracted from
mycelium with a mixture of acetone and 4% tartaric acid (1 : 1).
For quantitative analysis of individual alkaloids a fluorodensi-
tometric method was used (Pro~ek et al. 1977).
118 S. Mili6i6 et al. : C. purpurea growth and alkaloid biosynthesis

Total carbohydrates were estimated as glucose by the anthrone excreted into the medium. Excretion of alkaloids
method (Vahouny et al. 1960). almost stopped on the eighth day, when growth
Inorganic phosphate was measured by the method of Beren- velocity decreased to a value of about 0.25 g/1 per
blum and Chain (1938). hour, whereas intracellular alkaloids began to ac-
cumulate rapidly.
p H measurements were carried out with an electrode 465-35 K1 Following the composition of alkaloids, it was
(Ingold) and pH-meter M-7822 N (Intec, Stuttgart).
found that the percentage of ergotoxines in-
creased, while the LAD percentage decreased to-
wards the end of the fermentation (Table 1).
Results and discussion By feeding the culture with phosphate (0.09 g/
1) and amonoxalate (0.015 g/l) on the second day
As shown in Fig. 1, the fungal growth velocity of the fermentation (Fig. 2) we obtained higher
(dX/dt) reaches two maxima during Claviceps pur- fungal growth velocities during idiophase. This
purea batch fermentation: the first in trophophase resulted in higher fungal biomass and alkaloid
between the second and the third day, and the yield. In comparison with non-fed cultures some
second during idiophase, between the fifth and differences could be observed, not only in the dis-
the sixth day. The minimum growth velocity be- tribution of alkaloids, but also in their composi-
tween the third and fourth day coincided with the tion. In fed culture a higher extracellular but
pH minimum, phosphate exhaustion and the on- lower intracellular alkaloid content and a higher
set of alkaloid production. percentage of LAD were established at the end of
Such results indicate the phenomenon of the process (Table 2). A higher content of ergo-
diauxic growth, which we have already described cornine was also determined, as well as a higher
in our previous paper (So~i~ et al. 1985). ratio between ergocryptine a/fl isomers.
From the beginning of idiophase up to the A similar example of apparent interaction be-
maximum of growth velocity, alkaloids are mostly tween secondary metabolism and growth rate oc-

Table 1. Changes of ergotoxine and LAD content during Clavicepspurpurea L-17 batch fermentation

Day of fermentation 6 7 8 9 10 11 12 13 14

Ergotoxines (%) 60.8 62.5 64.2 64.4 66.8 67.6 68.7 70.1 71.4

LAD (%) 26.6 24.5 23.4 21.3 20.3 19.4 17.8 16.3 15.9

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Fig. 1. The relationship between growth velocity, pH, phos- Fig. 2. Alkaloid distribution, extracellular (x) and intracellular
phate uptake and extracellular (x) and intracellular (@) alka- ([] • ) , as a function of growth velocity pattern in C. purpurea
loid content in Claviceps purpurea batch fermentation fed (A) and not fed (B) batch fermentation
S. Mili6i6 et al. : C. purpurea growth and alkaloid biosynthesis 119

Table 2. Alkaloid content in fed and non fed C. purpurea 14-day-old cultures

Final Total Intracell. Extracell. Intracell. Intra- Extracell. Extra- Ergocornine/ Ergo-
biomass alk. alkaloid alkaloid ergotoxines cell. ergotoxines cell. Ergocryptine cryptine
(g/l) content content content (%) LAD (%) LAD (a/fl)
(mg/l) (mg/1) (mg/1) (%) (%)

Fed culture 77.6 1738 1028 710 80.1 4.4 46.0 51.4 1.29 3.24
Non ~ d culture 65.9 1626 1158 468 85.0 2.6 38.0 48.9 0.84 1.78

curs also in the production of gibberellin and bi-
kaverin by Gibberella fujikuroi (Bu'Lock et al.
1975).
The results we obtained indicate that biosyn-
thesis of different alkaloids may be favoured by
different growth velocities during idiophase of C.
purpurea batch fermentation. Formation of extra-
cellular alkaloids is favoured under higher rates
of growth, while that of intracellular alkaloids is
favoured under lower growth rates. We assume
that synthesis of D-lysergic acid in C. purpurea
fermentation must proceed throughout the idio-
phase, since no ergopeptines could otherwise be
formed. The key role of D-lysergic acid in peptide
alkaloid biosynthesis was observed and discussed
by Keller et al. (1980). It may be concluded that
LAD and D-lysergic acid biosynthesis could pro-
ceed in a wide range of fungal growth velocities,
while ergotoxine biosynthesis is more limited.
This conclusion also allows the differences in
growth and alkaloid biosynthesis obtained with
innocula of different magnitude (Table 3) to be
explained. We consider that cultures inoculated
with a small amount of inoculum, assuring higher
yields of cell material (Yx/s), grew during idio-
phase at a higher growth rate, but for a shorter
time. The consequences were low biomass and to-
tal alkaloid yield with higher LAD content and
lower ratio of intra- and extracellular alkaloids.
On the other hand, cultures inoculated with a
large inoculum, and with lower Yx/~, grew during
idiophase at a lower growth rate but for a longer
time. Large-inoculum cultures resulted in higher
biomass and alkaloid yield with higher ergotoxine
content and higher ratio between intra- and extra-
cellular alkaloids. The same correlation among
growth kinetics, yields of cell material and inocu-
lum size was established with Aspergillus oryzae
by Meyrath and McIntosh (1963).
Summarizing the results it can be claimed that
alkaloid yields in C. purpurea submerged batch
fermentation depend on time the fungus spends
growing in the idiophase, while the composition
of alkaloids and their distribution depends on the
absolute value of growth velocity during that peri-
od. These observations can be of importance for
improving submerged alkaloid production since,
by regulating growth velocity during idiophase, it
is possible to direct the production of desirable
alkaloids within the limits of strain biosynthetic
capabilities.
References
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Table 3. Effects of the inoculum size on the growth and alkaloid biosynthesis in C. purpurea submerged fermentation

Inoculum Final Yield Total alk. Intracell. Extracell. Ergo- LAD Ergocornine/
V/V % biomass factor content alkaloid alkaloid toxines (%) Ergocryptine
(g/l) (mg/1) content content (%)
(mg/l) (mg/t)

5 49.1 0.47 1053 423 630 30.7 52.1 1.44

10 67.3 0.43 1523 1011 512 57.0 25.4 1.20
20 72.8 0.37 1633 1173 460 63.4 16.2 1.12
120
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Received August 25, 1986/Revised April 14, 1987