Process Biochemistry 37 (2002) 1257 1262

www.elsevier.com/locate/procbio

Effect of aeration rate on the mycelial morphology and

exo-biopolymer production in Cordyceps militaris
Jong Pil Park a, Young Mi Kim a, Sang Woo Kim a, Hye Jin Hwang a,
Youn Jeung Cho a, Yong Se Lee b, Chi Hyun Song a, Jong Won Yun a,*
b

a
Department of Biotechnology, Taegu Uni6ersity, Kyungsan, Kyungbuk 712 -714, South Korea
Department of Natural Resources, Taegu Uni6ersity, Kyungsan, Kyungbuk 712 -714, South Korea

Received 23 October 2001; accepted 4 December 2001

Abstract
The influence of aeration rate on Cordyceps militaris morphology and exo-biopolymer production was investigated in a 5-l jar
fermentor. The mycelial morphology of C. militaris was characterized by image analysis, which included mean diameter,
circularity, roughness, and compactness of the pellets. Cells were observed to form mainly pellets during the entire culture period
irrespective of aeration conditions. There existed a notable variation in morphological parameters between the pellets grown on
different aeration conditions, by which exo-biopolymer production yields were correspondingly altered. The mean diameter and
compactness of the pellets indicated higher values at 2 vvm (volume of air per volume of culture per minute), which was closely
related to exo-biopolymer biosynthesis. The more compact pelleted form was favourable for exo-biopolymer production. Under
extremely low and high aeration conditions (e.g. 0.5 and 4 vvm), severe deformations of pellets (autolysis of core and shaving off
the outer hairy region) were observed at the later stages of fermentation associated with a decrease in morphological parameters.
2002 Elsevier Science Ltd. All rights reserved.
Keywords: Aeration rate; Cordyceps militaris; exo-biopolymers; Morphology; Submerged culture

1. Introduction
Mushrooms have been regarded as popular folk or
effective medicines used to treat human diseases such as
hepatis, hypertension, hypercholesterolemia, and gastric
cancer [15]. Both crude exo-biopolymers produced by
submerged culture of Cordyceps species and mycelial
biomass have recently been regarded as desired products with perceived health benefits [6]. Cordyceps militaris, belonging to the class ascomycetes, has received
special attention for medicinal purpose due to its various physiological activities [5,6].
Fungal morphology is an important parameter that
affects the rheological properties of the fermentation
broth, and control of the morphology is highly desired
in industrial fungal fermentation [7 11]. In general, two
* Corresponding author. Tel.: + 82-53-850-6556; fax: +82-53-8506559.
E-mail address: jwyun@taegu.ac.kr (J.W. Yun).

growth forms, the filamentous and the pelleted form,

can be observed in most fungal fermentations and the
pelleted form is usually less viscous than the filamentous form [12,13]. Pellets are characterized by the mycelia developing into stable, spherical aggregates
consisting of a more or less dense, branched and partially intertwined network of hyphae [14]. A number of
reports have been documented concerning factors influencing fungal morphology, rheology and production of
microbial exo-biopolymers (e.g. polysaccharides), major products from many fungi [15 19]. Amongst many
aspects of morphology, the effect of aeration has been
studied by a number of investigators [17,19 22]. By
varying the aeration rate, the shape of morphology
changed from filamentous to pellet, and vice versa [23].
The aim of this study was to characterize the morphology of C. militaris and thus to determine the
favourable mycelial form for exo-biopolymer production by varying the aeration rates in a 5-l batch
bioreactor.

0032-9592/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved.
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J.P. Park et al. / Process Biochemistry 37 (2002) 12571262

2. Materials and methods

2.1. Microorganism and media

C. militaris was kindly provided by Dr JM Sung,
Kangwon National University, Korea. The microorganism was maintained on potato dextrose agar (PDA)
plates and subcultured every month. Slants were incubated at 20 C for 7 days and then stored in a refrigerator. The seed culture was grown in a 250 ml flask
containing 50 ml of PMP medium at 20 C in a shake
incubator at 150 rpm for 5 days.

2.2. Inoculum preparation

C. militaris was initially grown on PDA medium in a
petridish, and transferred into the seed culture medium
by punching out from the agar plate culture with a
cutter [24]. Flask culture experiments were performed in
a 250 ml flask containing 50 ml of the media after
inoculating with 4% (v/v) of the seed culture.

2.3. Fermentation conditions

The medium composition for the fermentation was as
follows: 40 g sucrose, 10 g corn steep powder, 0.5 g
K2HPO4, 0.5 g KH2PO4, 0.5 g MgSO47H2O, 0.1 g
FeSO47H2O in 1-l distilled water [24]. The fermentation was carried out in a 5-l batch bioreactor (KOBIOTECH Co., Seoul, Korea) with a working volume of
3-l. The production medium was inoculated with 4%
(v/v) (: 0.4 g/l dry equivalent of cells) of the seed
culture and then cultivated for 10 days at 20 C. The
pH and agitation rates were controlled at 6.0 and 150
rpm, respectively. All experiments were performed in
triplicate to ensure the trends observed were
reproducible.

of crude exo-biopolymers was freezedried in a

lyophilizer and the weight of the polymer was estimated. For a quantitative measurement of sucrose, the
filtrate from membrane filtration (0.45 mm, Millipore)
was analyzed by high performance liquid chromatography, using an Aminex HPX-42C column (0.78 30 cm;
Bio-rad, USA) equipped with a refractive index detector (Shimadzu Co., Kyoto, Japan).

2.5. Characterization of the morphology

The morphological properties of the samples collected were evaluated using an image analyzer (Matrox
Electronic System, Canada) with software linked to a
light microscope (Olympus, USA) through a CCD camera. Samples were fixed with an equal volume of fixative (13 ml of 40% formaldehyde, 5 ml glacial acetic
acid, 200 ml of 50% ethanol). An aliquot (0.1 ml) of
each fixed sample was transferred to a slide, air dried,
and then stained with methylene blue (0.3 g of methylene blue, 30 ml of 95% ethanol in 100 ml water) [25].
For each sample, the morphology of 50 pellets was
characterized by measuring the area and perimeter of
the pellet core and the maximum diameter of the pellet.
Normally, a magnification of 100 was used. The morphology of the pellets was characterized by their mean
diameter, circularity, roughness and compactness. The
circularity or shape factor was estimated as the ratio of
the Fierets minimum diameter to the Fierets maximum diameter of the pellets or aggregates. The compactness was estimated as the ratio of the projected
area of the hyphae in a clump to the projected convex
area of that clump, the latter being the area after filling
internal voids and concavities in the clumps external
perimeter. In addition, the roughness (R) was measured
using the following equation: R=(pellet/aggregate
perimeter)2/(4p pellet area) [26].

2.4. Analytical methods

3. Results and discussion
The dry weight of mycelium was measured after
repeated washing (with distilled water) of the mycelial
pellet, obtained after the first centrifugation (10 000 g
for 10 min) and then dried at 90 C for 12 h, to
constant weight. This weight was compared to the total
weight obtained from the filtrates. To measure the
exo-biopolymer concentration, the samples collected at
various intervals from the bioreactor were centrifuged
at 10 000g for 15 min, and the supernatant was
filtered through a membrane filter (0.45 mm). The resulting culture filtrate was mixed with four times volume of absolute ethanol, stirred vigorously and left
overnight at 4 C. The precipitated exo-biopolymers
were centrifuged at 10 000g for 10 min and the
supernatant was discarded. The residue was re-precipitated with four times volume of ethanol, the precipitate

3.1. Morphological obser6ation under different aeration

rates
Pellet morphology has been cited as one of the key
factors determining fermentation productivity [8]. In
this study, the pellet morphology was characterized
with respect to pellet diameter, circularity, roughness,
and compactness of a pellet. Pellets were differentiated
from fungal fragments and clumps by their grayness
level. The amount of grayness, which was used as a
critical threshold level or cut-off, was previously obtained by analyzing many images. The pellets were
stained as described earlier, which were easy for determination of the core pellets and the outer hairy regions
accurately. Fig. 1 shows the typical morphological

J.P. Park et al. / Process Biochemistry 37 (2002) 12571262

changes in C. militaris cell during the entire fermentation period under various aeration conditions. The cells
were observed to form mainly pellets during the entire
culture period irrespective of aeration conditions. However, as the fermentation proceeded, the outer hairy
region of the pellets were shaved off and the core area
was reduced, resulting in a corresponding decrease in
pellet circularity and roughness, although the cell concentration remained the same in the stationary phase.
At the later stages of fermentation, typically after day 8
in this work, the pellets not only broke up but also the
mycelia lost rigidity and appeared somewhat withered
(Fig. 1). Furthermore, pellet autolysis was accelerated
at 0.5 vvm due to deletion of dissolved oxygen (starvation), consequently to form a looser mycelial clump
(Fig. 1A). Moreover, higher aeration (4 vvm) also
caused pellet autolysis because complete consumption
of substrate (sucrose) resulted from rapid mycelial
growth. The population of the free mycelia in the
fermentation medium was also least during the growth
phase, which indicated that pellets, rather than free
mycelia, were the more productive morphological forms
of C. militaris for optimal cell growth and exo-biopolymer production as described later. Cui et al. [27] reported that high DO tension produced denser pellets of
Aspergillus awamori, whereas weak and fluffy pellets
were formed under very low DO tension.

3.2. Characterization of mycelial morphology

Morphological properties of a filamentous fungus
play an important role in their metabolism during
fermentation processes [7 10,28]. Fig. 2 shows the
mean diameter, circularity, roughness, and compactness

1259

of the pellets during submerged culture of C. militaris

under different aeration conditions. There was a significant variance in the morphological parameters between
the pellets grown under different aeration conditions.
The pellet diameter increased rapidly from the first day
to the end of fermentation at an aeration rate of 2 vvm.
However, pellet sizes decreased at a low and a high
aeration conditions after day 6, which indicated that
pellet lyses occurred due to either higher oxygen starvation (0.5 vvm) or rapid depletion of substrate associated
with facilitated oxygen supply into core of the pellets (4
vvm). The maximum value of the pellet mean diameter
grown at 0.5, 2 and 4 vvm were about 0.53, 0.71 and
0.58 mm, respectively. The circularity and roughness
observed at aeration rates of 0.5 and 4 vvm increased
from the early stage of fermentation (till day 6) and
thereafter slowly decreased as in previous morphological observations (Fig. 2B and C). This result was probably caused by loosening of the pellet cores, after which
the pellets rapidly broke up into hyphal fragments and
flocks (Fig. 1A and C). In other words, the outer hairy
region of the pellets were shaved off, which resulted in
corresponding decrease in pellet roughness and pellet
diameter although the cell concentration remained the
same in the stationary phase. Since shaving off hyphae
is a function of hydrodynamic forces, minimum roughness was observed under high aeration condition (4
vvm). The decrease in pellet roughness can be explained
by severe hyphal fragmentation as a result of the reduction of the hyphal tensile strength due to a successive
vacuole formation [29]. Taking into account that circularity mean a shape factor describing the deviation of
the pellet image from a true circle, and roughness mean
the irregularity of the perimeter of pellet, compact

Fig. 1. Morphological changes in C. militaris in a 5-l batch reactor. 2 10 d mean fermentation period in day.

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J.P. Park et al. / Process Biochemistry 37 (2002) 12571262

Fig. 2. (A) Mean diameter, (B) circularity, (C) roughness, and (D) compactness of C. militaris pellets growing in a 5-l batch reactor at the aeration
rates of 0.5 vvm (), 2 vvm (
), and 4 vvm ().

pellet formation with delaying pellet deformation was

favourable at an aeration rate of 2 vvm. This agreed
with the result that compactness (fullness) of the pellets
indicated a maximum and remained nearly constant at
2 vvm (Fig. 2D). Riley et al. [26] have pointed out that
compactness and roughness of Penicillium chrysogenum
pellets were kept nearly constant during the entire
period of fermentation. In a review article by Paul and
Thomas [30], compactness and circularity of Aspergillus
niger pellets decreased slowly while the core area
sharply increased during the exponential growth of
mycelia. Olsvik et al. [31] have reported similar result to
the present findings; i.e. A. niger pellets showed decreased roughness as the DO level increased, whereas
maximum compactness was observed at an intermediate DO level examined (12%). Low roughness of the
pellets observed at a high oxygen level in the vessel
might imply some effect on hyphal hyphal interaction
within the mycelial clump.

3.3. Effect of aeration rate on mycelial growth and

exo-biopolymer production
In a previous paper [24], it was reported that sucrose
was the most suitable carbon source for both mycelial
growth and exo-biopolymer production in C. militaris.
Fig. 3A shows the time profile of sucrose consumption
at different aeration rates. The concentration of resid-

ual sucrose sharply decreased during the fermentation

with corresponding increase in biomass and exo-biopolymer production. Almost complete sugar depletion
was observed for the culture grown at an aeration rates
of 4 vvm, whereas 17 and 10 g/l sucrose remained in the
fermentation broth in cultures grown at aeration rates
of 0.5 and 2 vvm for the same fermentation period.
High aeration rate of 4 vvm clearly resulted in substantially increased mycelial growth (Fig. 3B). In cultures under the examined aeration rates (0.5, 2 and 4
vvm), the maximum mycelial biomass yield increased
up to 5, 8, and 8 days, respectively, and then remained
constant during the later stages of fermentation. Maximum exo-biopolymer production (14.5 g/l) was
achieved at 2 vvm, whereas a further increase in aeration rate at values over 2 vvm or below, resulted in a
decrease in exo-biopolymer production (Fig. 3C).
It is noteworthy that the combination of dissolved
oxygen concentration with favourable morphology
played an important role in higher exo-biopolymer
production. The DO level at an aeration rate of 2 vvm
was reduced from 100% saturation at the beginning of
fermentation to around 15% saturation at days 23,
thereafter high DO level (76%) was maintained
throughout the fermentation, allowing the pellets to
show a more compact form (Fig. 3). These results are
also in good agreement with those of other investigators [32]. Gibbs and Seviour [16] and Wecker and

J.P. Park et al. / Process Biochemistry 37 (2002) 12571262

Onken [19] have reported that the inhibitory effect of

high dissolved oxygen concentration was observed on
polysaccharide production in Aureobasidium pullulans.
Therefore, the decrease in exo-biopolymer production
at aeration rates higher than 2 vvm or below was due
either to the change in morphology of the microorganism during fermentation or substantial cell damage or
change in a mycelial regulatory mechanism [33] (Fig.
3A and C).

4. Conclusion
From the above results, a critical conclusion was
derived that the aeration rate was an important factor
for exo-biopolymer production controlling the growth
of C. militaris in a more compact pelleted form. Mor-

Fig. 3. Time profiles of (A) sucrose consumption, (B) mycelial

growth, and (C) exo-biopolymer production in C. militaris using a 5-l
batch reactor at various aeration rates of 0.5 vvm (), 2 vvm (
), and
4 vvm ().

1261

phological parameters may depend on the fungal species, the growth medium or the physical environment
within the culture vessel. Extensive studies on the exobiopolymers, including molecular characterization and
structure identification, are ongoing in this laboratory.

Acknowledgements
This work was supported by the RRC program of
MOST and KOSEA.

References
[1] Kiho T, Hui J, Yamane A, Ukai S. Polysaccharides in fungi.
XXXII. Hypoglycemic activity and chemical properties of a
polysaccharide from the cultural mycelium of Cordyceps sinensis.
Biol Pharm Bull 1993;16:1291 3.
[2] Shiao MS, Wang ZN, Lin LJ, Lien JY, Wang JJ. Profiles of
nucleotides and nitrogen bases in Chinese medicinal fungus
Cordyceps sinensis and related species. Bot Bull Acad Sin
1994;35:261 7.
[3] Sone Y, Okuda R, Wada N. Structures and antitumor activities
of the polysaccharides isolated from fruiting body and the
growing culture of mycelium of Ganoderma lucidum. Agric Biol
Chem 1985;49:2641 53.
[4] Lee J, Chung C, Jeong H, Lee K. Anticomplementary and
antitumor activities of the alkali extract from the mycelia of
Lentinus edodes IY 105. Kor J Appl Microbiol Biotechnol
1990;18:571 7.
[5] Song CH, Yang BK, Ra KS, Shon DH, Park EJ, Go GI, Kim
YH. Hepatoprotective effect of extracellular polymer produced
by submerged culture of Ganoderma lucidum WK-003. J Microbiol Biotechnol 1998;8:277 9.
[6] Kuo YC, Tasi WJ, Shiao MS, Chen CF, Lin CY. Cordyceps
sinensis as an immunomodulatory agent. Am J Chin Med
1996;24:111 25.
[7] Charles M. Technical aspects of the rheological properties of
microbial cultures. Adv Biochem Eng 1978;8:1 62.
[8] Metz B, Kossen NWF, van Suijdam JC. The rheology of mold
suspensions. Adv Biochem Eng 1979;11:104 56.
[9] Tucker KG, Thomas CR. Effect of biomass concentration and
morphology on the rheological parameters of Penicillium chrysogenum fermentation borths. Trans Inst Chem Eng 1993;71:111
7.
[10] Moreira MT, Sanroman A, Feijoo G, Lema JM. Control of
pellet morphology of filamentous fungi in fluidized bed bioreactors by means of a pulsing flow. Application to Aspergillus niger
and Phanerochate chrysosporium. Enzyme Microb Technol
1996;19:261 6.
[11] Jimenez-Tobom GA, Penninckz MJ, Lejeune R. The relationship
between pellet size and production of Mn (II) peroxidase by
Phanerochate chrysosporium in submerged culture. Enzyme Microb Technol 1997;21:537 42.
[12] Berovic M, Cimerman A, Steiner W, Koloni T. Submerged citric
acid fermentation: rheological properties of Aspersillus niger
broth in a stirred tank reactor. Appl Microbiol Biotechnol
1991;34:579 81.
[13] Sinha J, Bae JT, Park JP, Kim KH, Song CH, Yun JW. Changes
in morphology of Paecilomyces japonica and their effect on broth
rheology during production of exo-biopolymers. Appl Microbiol
Biotechnol 2001;56:88 92.

1262

J.P. Park et al. / Process Biochemistry 37 (2002) 12571262

[14] Berovic M, Cimerman A. Redox potential in submerged citric

acid fermentation. Appl Microbiol Biotechnol 1982;16:185 8.
[15] Bae JT, Sinha J, Park JP, Song CH, Yun JW. Optimization of
submerged culture conditions for exo-biopolymer production by
Paecilomyces japonica. J Microbiol Biotechnol 2000;10:482 7.
[16] Gibbs PA, Seviour RJ. Does the agitation rate and/or oxygen
saturation influence exopolysaccharide production by Aureobasidium pullulans in batch culture? Appl Microbiol Biotechnol
1996;46:503 10.
[17] Stasinopoulos SJ, Seviour RJ. Exopolysaccharide production by
Acremonium persicinum in stirred-tank and air-lift fermentors.
Appl Microbiol Biotechnol 1992;36:465 8.
[18] Dosoretz CG, Chen AHC, Grethlein HE. Effect of oxygenation
conditions on submerged cultures of Phanerochaete chrsosporium. Appl Microbiol Biotechnol 1990;34:131 7.
[19] Wecker A, Onken U. Influence of dissolved oxygen concentration and shear rate on the production of pullulan by Aureobasidium pullulans. Biotechnol Lett 1991;13:155 60.
[20] Elibol M, Ozer D. Influence of oxygen transfer on lipase production by Rhizopus arrhizus. Process Biochem 2000;36:325 9.
[21] Veglio F, Beolchini F, Ubaldini S. Empirical models for oxygen
mass transfer: a comparigon between shake flask and lab-scale
fermentor and application to manganiferous ore bioleaching.
Process Biochem 1998;33:367 76.
[22] Kamilakis EG, Allen DG. Cultivating filamentous microorganisms in a cyclone bioreactor: the influence of pumping on cell
morphology. Process Biochem 1995;30:353 60.
[23] van Suijdam JC, Metz B. Influence of engineering variables upon
the morphology of filamentous molds. Biotechnol Bioeng
1981;23:111 48.
[24] Park JP, Kim SW, Hwang HJ, Yun JW. Optimization of submerged culture conditions for the mycelial growth and exo-bio-

[25]

[26]

[27]

[28]

[29]

[30]
[31]

[32]

[33]

polymer production by Cordyceps militaris. Lett Appl Microbiol

2001;32:1 6.
Packer HL, Thomas CR. Morphological measurements on
filamentous microorganisms by fully automatic image analysis.
Biotechnol Bioeng 1990;35:870 81.
Riley GL, Tucker KG, Paul GC, Thomas CR. Effect of biomass
concentration and mycelial morphology on fermentation broth
rheology. Biotechnol Bioeng 2000;68:160 72.
Cui YQ, van der Lans RGJM, Luyben KChAM. Effects of
dissolved oxygen tension and mechanical forces on fungal morphology in submerged fermentation. Biotechnol Bioeng
1998;57:409 19.
Nielsen J, Johansen CL, Jacobsen M, Krabben P, Villadsen J.
Pellet formation and fragmentation in submerged cultures of
Penicillium chrysogenum and its relation to penicillin production.
Biotechnol Prog 1995;11:93 8.
Cui YQ, Okkerse WJ, van der lans RGJM, Luyben KChAM.
Modeling and measurements of fungal growth and morphology
in submerged fermentations. Biotechnol Bioeng 1998;60:216 29.
Paul GC, Thomas CR. Characteristics of mycelial morphology
using image analysis. Adv Biochem Eng 1998;60:1 59.
Olsvik E, Tucker KG, Thomas CR, Kristiansen. Correlation of
Aspergillus niger broth rheological properties with biomass concentration and the shape of mycelial aggregates. Biotechnol
Bioeng 1993;42:1046 52.
Park YS, Ohta N, Okabe M. Effect of dissolved oxygen concentration and impeller tip speed on itaconic acid production by
Aspersillus terreus. Biotechnol Lett 1993;15:583 6.
Kubicek CP, Zehentgruber O, El-Kalak H, Rohr M. Regulation
of citric acid production by oxygen: effect of dessolved oxygen
tension on adenylate levels and respiration in Aspersillus niger.
Eur J Appl Microbiol Biotechnol 1980;28/29:667 84.