Journal of General Microbiology (1988), 134, 2685-2692.

Printed in Great Britain

2685

Microcycle Conidiation in Submerged Cultures of Penicillium cyclopium

Attained without Temperature Changes
BY JAROSLAV P A Z O U T ~ *A N D PETER S C H R ~ D E R ~
IInstitute of Microbiology, Czechoslovak Academy of Sciences, Videliska 270, 142 20 Prague 4,
Czechoslovakia
Institut fur Medizinische Mikrobiologie und Epidemiologie der Martin-Luther- Universitat,
Leninallee 6, 402 Halle/Saale, GDR
(Received 12 April 1988; revised 14 June 1988)

Microcycle conidiation in shaken cultures of Penicillium cyclopium M 79 was induced at 24 "C

without any shock treatment. The occurrence of a microcycle depended on the presence of an
organic acid (especially glutamic acid) in combination with glucose, low phosphate
concentration, light and sufficientaeration. Absence of glucose and/or lowered aeration evoked
vegetative growth. A synchronous and homogeneous microcycle required a certain relationship
between the number of inoculated conidia and the concentration of the organic acid in the
medium; the optimum was at 0.08 nmol acid per conidium. Higher values stimulated normal
vegetative growth. A shortage or absence of the organic acid led to an atypical growth. The
effect of organic acid can be partially replaced by addition of 2% (w/v) CaC03. Addition of
NHI in concentrations higher than 6mM to cultures with glutamate or glutamine evoked
vegetative growth.

INTRODUCTION

Microcycle conidiation is a shortened developmental cycle in which conidiogenesis starts

immediately after spore germination and proceeds without mycelial growth. Microcyclic
conidiation has been described in a number of asexual fungi (i.e. Aspergillus niger, Paecilomyces
uarioti, Penicillium urticae, Neurospora crassa, Penicillium digitatum) in submerged cultures
(Anderson & Smith, 1971;Anderson et al., 1978; Cortat & Turian, 1974; Sekiguchi et al., 1975)
and in surface cultures (Zeidler & Margalith, 1973). Until now, a supraoptimal temperature was
mentioned as a common feature for inhibiting apical growth and inducing microcycle
conidiation. A typical phenomenon of the microcycle is the so-called spherical growth of the
inoculated conidia following conidial swelling (hydration).
Sekiguchi et al. (1975) found an increase in the number of cell organelles and changes in the
character of the cell wall during this period. Duncan et al. (1978) found a maximum rate of
protein synthesis in the first third and of RNA accumulation in the first half of the incubation at
increased temperature, i.e. at the beginning of the spherical growth. Further microcycle
differentiation required neither protein synthesis nor RNA synthesis de nouo.
Our study reports the induction of the microcycle in submerged cultures of Penicillium
cyclopium by specificcomposition of the incubation medium which gives rise to spherical growth
of conidia at optimal cultivation temperature.
METHODS

Strain and cultivation conditions. Penicillium cyclopium Westling strain M 79 from the collection of this Institute
was cultivated for 3 d on Petri dishes with malt agar at 24 "C (Paiout et al., 1982). Sporulated cultures were then
kept for 11 d at 4 "C.Dry mature conidia were scraped off the surface of the cultures and suspended in saline. The
suspension was used to inoculate media (to a final concentration of 3-6 x lo5 conidia per ml unless otherwise
stated) of the SM basic salt composition CaC12.6H20, 0.4 g; KH2P04, 0.125 g; MgS04.7H20, 0.15 g;

0001-4834 0 1988 SGM

2686

J . P A ~ O U TA N D P . SCHRODER

FeS04.7H20,0.005 g; KCl, 0.5 g; ZnS04.7H20, 0.0155 g; distilled H 2 0 to 1 litre, pH 5.6 before sterilization
(Schroder, 1978)with an addition of 34 mM-L-glUtamiC acid and 17 mM-Na2S04or 34 m~-2-oxoglutaricacid with
17 ~ M - ( N H ~ ) ~ASsecond
O ~ . carbon source was glucose at a concentration of 5.55 or 275.5 m ~Shaken
.
cultures
were cultivated in 300 ml Erlenmeyer flasks containing 100 ml of medium, at 24 C on a rotary shaker (frequency
4.17 Hz, excentricity 5.5 cm). Artificial pH regulation was done in cultures growing in a 1 litre glass fermenter
containing 600 ml of medium with an aeration rate of 300 ml air min- and magnetic stirring (frequency 10 Hz).
The pH was adjusted with 0.5M-NaOH or 0.25M-H2S04 (Schriider, 1982). All cultures were illuminated
throughout the cultivation from a distance of 90 cm by a 40 W tube (Tesla) giving off a bluish light (Schlette, 1980;
Pakut et al., 1982). Cultivations were run in triplicate and each experiment was repeated three times.
Analytical methods. Glutamic acid was determined colorimetrically by the ninhydrin reaction (Moore & Stein,
1954); the cultivation fluid was checked chromatographically for the presence of the other amino acids.
2-oxoglutarate and oxaloacetate were determined together according to the general procedure of Friedman &
Haugen (1943). The reaction with hydrazine was prolonged to 25 min and sample absorbance was measured at
420 nm. The ratio of the two acids was always determined chromatographically before the actual quantitative
assay (Barker & Mapson, 1953). NHX was determined by commercial Nessler reagent. Glucose was assayed using
the Bio-LA-Test glucose enzymically, (Lachema, Brno, Czechoslovakia), based on the glucose oxidase plus
peroxidase reactions. Utilization of glucose was measured by 4C02evolution from universally labelled glucose
fed to shaken cultures (Godfrey & Snyder, 1962). The number of conidia was determined by using an
haemocytometer .
RESULTS

Optimum conditionsfor microcycle induction

The induction of the microcycle comprises inhibition of apical growth of the germ-tube(s) and
extension of the stage of conidial swelling (mainly hydration and nutrient intake) by so-called
spherical growth. Spherical growth is accompanied by active growth of biomass (Duncan et al.,
1978) and, after its termination, a conidiophore is formed instead of a germ-tube. The first cell
division takes place only at the moment of formation of the first new conidium. Spherical growth
is conveniently induced by incubating the spore suspension at a supraoptimal temperature.
In our cultures, the microcycle was observed after inoculation of mature conidia into the
synthetic SM medium containing glucose plus glutamate (or 2-oxoglutarate). Cultures attaining
the microcycle had to be intensively agitated on a rotary shaker at a frequency of 4.17 Hz. On a
reciprocal shaker with a frequency of 1-67Hz, spherical growth did not occur, the conidiation
onset was accelerated, but no true microcycle took place.
Swelling of conidia lasted 3-4 h, and spherical growth lasted a further 3-4 h; the germination
of the resulting giant cells thus took place after 6-8 h. Fully developed sporophores and conidia
appeared 24-36 h after inoculation (Fig. 1). In high glucose cultures, the germination of newly
formed spores took place 40 h after inoculation. In low glucose cultures this was not observed.
The induction of the spherical growth was crucially affected by the medium composition,
agitation intensity and light. Another important factor for the triggering of the microcycle was
the pH change from the initial pH 5.6 to 4.5-4-3 during the first 8-12 h of cultivation, probably
caused by a faster incorporation of NH,+ or amino group of glutamate as compared with the
utilization of the organic acid (Figs 2 and 3). Buffering of the medium at pH 5.6 stopped culture
development at the stage of an early germination of giant cells; the vegetative growth began only
15 h after inoculation and conidia were rarely formed 24 h after inoculation.
Cultures inoculated with 6-3 x lo4 or fewer conidia ml-1 were incapable of lowering the pH
value of the medium and the giant cells grew vegetatively after germination. An artificially
induced pH drop, however, caused no initiation of sporophores on the germ-tube; the vegetative
growth continued and conidiation took place about 24 h after inoculation.
Nutrient utilization during microcycle
The higher the glucose concentration in the medium, the more extensive was the conidiation.
The glucose concentrations tested were 0.55, 1.11,2.78,5-55, 11.1,27.8,55.5, 111 and 277.5 m~
while the glutamate concentration was kept constant (34 mM). The concentrations 0.55-1.1 1 mM
were the lowest to still initiate a microcyclic conidiation; however, only one daughter conidium
was formed. In further experiments only 5.55 or 277.5 mM glucose was used.

Submerged microcycle of P . cyclopium

2687

Fig. 1. Microcycle in a shaken culture of Penicillium cyclopium in medium with glucose (275.5 mM) and
2-oxoglutarate. Bar, 4 pm.

NH:

%
8
c

tra

I------

Or-

8 'Ot
c

PH

'
Glc

2o

NH:

DM

PH

0
'

12

24

36
48
Time (h)

60

2-OG
72
Time (h)

Fig. 2. Growth, nutrient utilization and pH in microcyclic cultures of Penicilliurn cyclopium grown in a
medium with 2-oxoglutarate. (a) Low glucose (5.55 mM) culture. (b) High glucose (275.5 mM) culture.
Glc, Glucose concentration (g 1-* ); 2-OG, 2-oxoglutarate concentration (g PI); DM,dry biomass
concentration (g 1-l). NHt concentrations are m~ values.

Glucose utilization took place together with the emergence of the germ-tube (6-8 h post
inoculation).Up to that time, no 14C02was produced from [U-14C]glucose.The consumption of
organic acid, however, started immediately, but its course during cultivation depended on
glucose concentration. The same held true for nitrogen utilization. Utilization of the nitrogen
source until the 12th hour of cultivation was identical in media with low and high glucose

2688

J . P A ~ O U TA N D P . SCHRODER

40

20
NH,+

T,

Glu

L,

I-

0'

12

24

36
48
Time (h)

60

72

"0

12

24

36
48
Time (h)

60

72

Fig. 3. Growth, nutrient utilization, pH and changes in 2-oxoglutarate and NHt levels in microcyclic
cultures of Penicillium cyclopium grown in a medium with glutamate. (a)Low glucose (5.55 mM) culture.
(6) High glucose (275.5 mM) culture. Glu, Glutamate concentration (a
All
).
other abbreviations and
symbols are as in Fig. 2.

content. Differences in utilization of NHf and in the behaviour of the whole culture after 12 h
cultivation between a low glucose and a high glucose culture were due to glucose exhaustion from
the former culture, which took place approximately in the 18th hour of cultivation.
In low glucose cultures with 2-oxoglutarate the content of NH,+ increased, probably due to
autolysis after 24 h of cultivation (Fig. 2a). In high glucose cultures NH,+ utilization slowed
down after about 24 h and the consumption of 2-oxoglutarate stopped. The residual NH,+level
was exhausted at the stage of germination of newly formed spores together with 2-oxoglutarate
(Fig. 26).
When glutamate was used (Fig. 3a) in low glucose cultures, 2-oxoglutarate and traces of
oxaloacetate appeared around the 12th hour after inoculation, both acids being again exhausted
before the 24th hour. The culture development then stopped and NH,+ from autolysed cells
appeared in the medium. The concentration of NHQ after 72 h of cultivation reached 10-15
mM. In high glucose cultures (Fig. 3 b) no NHQ appeared. Glutamate utilization had two phases
like the consumption of NH,+;2-oxoglutarate and traces of oxaloacetate appeared here between
the 6th and 20th hour of cultivation. No other amino and/or organic acids were found in culture
medium.
The pH of cultures with low glucose content dropped and then rose to pH 9.3 (Figs 2a and 3a),
due to organic acid utilization. After the formation of the usual one to three conidia per
sporophore, further differentiation was arrested and the conidia were not released into the
medium. Artificial buffering in a range of pH 4.6-6.0 caused an elongation of the conidial chain
to three to five conidia. In high glucose cultures the pH did not rise above 7.5 (Figs 2b and 3 b)
and microcycle conidiation proceeded undisturbed.

2689

Submerged microcycle of P . cyclopium

Table 1. Efect of the initial conidialglutamate ratio on microcycle intensity

V, mycelialgrowth; A, atypical thickened hyphae; F, microcyclein some parallel cultures, A in others;
M, microcycleand vegetative growth mixed; 1, less than 100 newly formed conidia per initial spore; 2,
100-500; 3, 500-2000; 4, more than 2000.

Type of growth
No. of
conidia ml-l
5.6 x 103
8.4 x 103
1-3 x 104
1.9 x 104
2.8 x 104

4.2 x 104

6.3 x 104
9.1 x 104
1.4 x 105
2.1
3-1
4.7
7.0

x
x
x
x
1.0 x
1.5 x
2.4 x

105
105
105

105
106
lo6
lo6

3.4

Glutamate concn ( m ~ )
6.8

17

34

51

2
3
4
4
2
A

A
A

The difference between nutrient utilization in high and low glucose cultures was due to
secondary germination of newly formed conidia. We suppose that the pH rise inhibiting further
development of conidia in low glucose cultures was prevented in high glucose cultures by a
transient arrest of 2-oxoglutarate or glutamate utilization.
Influence of medium changes
Excessively high or low concentrations of glutamate and inoculated spores are unfavourable
for triggering the microcycle. Optimum glutamate concentration was about 0.08 nmol per
conidium. Higher values caused induction of normal mycelial growth while lower levels evoked
atypical vegetative growth similar to that found in glutamate-free cultures. The glucose
concentration in this experiment was 277.5 mM ;however, lower concentrations (27.8, 55.5 and
111 mM) changed the biomass yield of the cultures but not their morphology. The effect of
glutamate concentration and inoculum size on the quality of the microcycle is shown in Table 1.
Cultures growing in a medium with 2-oxoglutarate or glutamate without glucose formed
conidia only after mycelial growth. Glucose medium plus 17 r n ~ - ( NH, ) ~ s0without
,
organic
acid(s) gave rise to atypical vegetative growth with thickened hyphae (Fig. 4). No conidiation
was observed and pH dropped to 1.8-2-0. Organic acids can be partially substituted by addition
of 2% CaC03 to the glucose medium. The culture maintained a stable pH of 5.8, conidial
swelling before germination was lower, the germ-tubes were longer and the microcycle appeared
only in conidia which developed in clusters around the CaC03 particles. Individually
germinating conidia were retarded in their development and the microcycle did not occur.
NH,+in concentrations above 6 m~ prevented the microcycle in cultures with glutamate or
glutamine (Table 2). In cultures with 2-oxoglutarate NH,+ increased the yield of conidia in
concentrations up to 51 mM. Glutamine was a less suitable substrate for microcycle than
glutamate.
Properties of newly formed conidia
Conidia from microcyclic high glucose cultures germinated at almost 100%frequency and, on
transfer into fresh SM medium in the 36th hour of cultivation, were capable of both microcyclic
and normal development. If left in the original culture, they germinated partially but a second

J . P A ~ O U TA N D P. SCHRODER

2690

Fig. 4. Shaken cultures of PeniciZZium cyclopium growing on only glucose (22.2 mM) medium (24 h). Bar,
4 pm.

Table 2. Eflect of N H i on the microcycle intensity in cultures with 2-oxoglutarate, glutamate and
glutamine
Substrate concn
(34 mM)

N H t concn
(mM)

Newly formed conidia

per one initial spore

2-Oxoglutarate

17
34
51
68
0
3.1
6.1
9.1
12.1 and higher
0
3.1
6.1
9.1
12.1 and higher

100
500-2000
2000
500-2000
2000
2000
500-2000
100-500
Vegetative growth
500-2000
100-500
100-500
Mixed growth
Vegetative growth

Glutamate

Glutamine

pH drop and exhaustion of nitrogen source led to the formation of a nearly sterile mycelium with
thin, slightly branched hyphae and isolated conidiophores. Conidia from low glucose cultures
after 36 h of cultivation had germinated at only 10% frequency and those which germinated
gave rise, on transfer, only to mycelial growth, not to the microcycle. When left in the original
culture, they lysed. After 72 h cultivation their germinating ability decreased to 0.5%. The
number of microcyclic conidia formed in a low glucose medium was approximately 30 per
original conidium whereas in high glucose medium it was up to 3000.
Culture pigmentation
Simultaneously with the appearance of first conidia (12th hour) the microcyclic cultures
exhibited a pink-violet tint of the medium; the intensity of the tint increased with the number of
conidia formed. In high glucose cultures the increasing intensity of the tint was accompanied by

Submerged microcycle of P . cyclopium

269 1

a shift in absorption maximum from 540 nm to 620 nm. Later, the colour was superseded by a
green conidial pigment. In low glucose cultures the shift of the maximum was hardly detectable;
after 72 h only 560 nm was reached, and the conidia remained hyaline. The pink-violet colour
was found in isolated cases also in normally differentiating cultures with a sufficient degree of
synchrony and closely preceded conidiation.
Attempts at isolating the pigment included concentration of the culture medium at 24 "C on a
rotary vacuum evaporator; the violet colour changed gradually to green. The change in the
absorption spectrum depended on the concentration of pigment and probably also on the
concentration of salts in the solution.

DISCUSSION

Most sporulating micro-organisms have a tendency to microcycle formation. However, a

distinction should be made between the occurrence and the quality of the microcycle. Penicillium
strains with high viability, high germinating ability of spores and high spore homogeneity also
form a homogeneous microcycle in terms of synchronous germination. Strains with lower
germinating ability more often form a mixture of normally differentiated mycelium and a
microcycle. In these strains a homogeneous microcycle may be achieved by a heat shock
(unpublished). In our case apical growth was inhibited solely by medium composition.
Anderson & Smith (1971) and Anderson et al. (1978) confirmed the crucial significance of
glutamate together with temperature shock to induce the microcycle in Aspergillus niger and
Penicillium varioti. Zeidler & Margalith (1973) found an asynchronous microcycle in surface
cultures of P . digitatum with glutamate and serine. We assume that the significance of glutamate
or 2-oxoglutarate lies in the inhibition of glucose utilization and Stimulation of oxidative
metabolism (Krebs cycle). The microcycle in P . cyclopium required a certain proportion of
glutamate concentration to the number of conidia. Too few inoculated spores led to normal
vegetative growth while excessive numbers caused atypical growth similar to that found in
cultures growing on glucose only. Only the correct ratio ensured an optimum course of pH
changes in the culture (unpublished), (Schriider & Schlette, 1978). When these acids were
replaced by CaC03, the conidia growing in clusters also formed the microcycle. We assume that
the effect of the suspended carbonate was due to the buffering of an intensively glycolysing
culture for an interval sufficient for the onset of oxidative metabolism to take place. There may
also be a stimulating effect of Ca2+. A prerequisite of a microcycle conidiation and of
conidiation generally was the presence of light and a sufficiently low initial concentration of
phosphate in the medium (Schlette, 1980; Paiout et al., 1982).
The amount of glucose in the medium affected the intensity of the microcycle, i.e. the number
of sporophores and newly formed conidia per primary spore. The minimum glucose
concentration which ensured the formation of at least one daughter conidium by the microcycle
was 0.1-0.2 g 1-l. The wide range of effective glucose concentrations excludes the effect of
catabolite repression on microcycle induction. In addition, glucose was not utilized during the
first 8 h of cultivation but its presence was necessary for the microcycle induction.
We suppose that glutamate induces the microcycle in P . cyclopium only after degradation to
2-oxoglutarate. Dubois et al. (1977) found a repression of glutamate dehydrogenase (NAD)
by NH,+ and by glutamine. In our experiments, NH,+ added to the medium with glutamate
or glutamine prevented the microcycle and evoked vegetative growth. We assume that
2-oxoglutarate is necessary for the microcycle as a stimulator of the Krebs cycle; however,
glutamate gave better conidial yield due to its wider utilizability in the cell.
The pink-violet pigment which was discovered because of the highly synchronized
microcyclic conidiation, perhaps represents a soluble precursor of the mature conidial pigment
or the pigment itself, partially dissolved.
The microcycle of submerged cultures of P . cyclopium has key points with specific
requirements : spherical growth, requiring intensive aeration and the presence of glucose plus
glutamate or 2-oxoglutarate in the medium; initiation of phialide and conidia formation, which

2692

J . P A ~ O U TA N D P. SCHRODER

depends on a pH drop in the culture; conidial maturation (defined here as the achievement of full
germinating ability and green colour), requiring also a sufficient initial concentration of glucose
(more than 10 g 1-l) in the medium. These three stages can be taken as a regulation sequence in
which any subsequent step depends on combination of the preceding step and an environmental
impulse.
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