Journal of Applied Microbiology 2003, 94, 120126

Optimization of submerged culture process for the production

of mycelial biomass and exo-polysaccharides by Cordyceps
militaris C738
S.-W. Kim1, H.-J. Hwang1, C.-P. Xu1, J.-M. Sung2, J.-W. Choi3 and J.-W. Yun1
1

Department of Biotechnology, Taegu University, Kyungsan, Kyungbuk 712-714, 2Department of Environmental Biology, Kangwon
National University, Kangwon 200-701 and 3Department of Natural Resources, Taegu University, Kyungsan, Kyungbuk 712-714, Korea

2002 163: received 17 April 2002, revised 24 June 2002 and accepted 7 July 2002

ABSTRACT
S . - W . K I M , H . - J . H W A N G , C . - P . X U , J . - M . S U N G , J . - W . C H O I A N D J . - W . Y U N . 2003.

Aims: The objective of the present study was to determine the optimal culture conditions for mycelial biomass and
exo-polysaccharide (EPS) by Cordyceps militaris C738 in submerged culture.
Methods and Results: The optimal temperatures for mycelial biomass and EPS production were 20C and 25C,
respectively, and corresponding optimal initial pHs were found to be 9 and 6, respectively. The suggested medium
composition for EPS production was as follows: 6% (w v) sucrose, 1% (w v) polypeptone, and 005% (w v)
K2HPO4. The influence of pH on the fermentation broth rheology, morphology and EPS production of C. militaris
C738 was carried out in a 5-l stirred-tank fermenter. The morphological properties were comparatively
characterized by pellet roughness and compactness by use of image analyser between the culture conditions with and
without pH control. The roughness and compactness of the pellets indicated higher values at pH-stat culture
(pH 60), suggesting that larger and more compact pellets were desirable for polysaccharide production (091 g g)1
cell d)1).
Conclusions: Under the optimized culture conditions (with pH control at 6), the maximum concentration of
biomass and EPS were 127 g l)1 and 73 g l)1, respectively, in a 5-l stirred-tank fermenter.
Significance and Impact of the Study: The critical effect of pH on fungal morphology and rheology presented in
this study can be widely applied to other mushroom fermentation processes.
Keywords: compactness, exo-polysaccharide, mycelial biomass, rheology, roughness.

INTRODUCTION
The genus Cordyceps (clavicipitaceae), a known group of
1 entomopathogenic fungi, form fruiting bodies in their
insect hosts, and 750 species in the genus have been
recognised (Sung 1996). Several Cordyceps species have
been used in traditional medicine in China, Japan, and
Korea (Sung 1996; Yu et al. 2001). These were very
difficult to collect because their sizes were very small and
their growth was restricted to a specific area. Recently
mass production of these strains through artificial cultivaCorrespondence to: Prof Jong Won Yun, Department of Biotechnology, Taegu
University, Kyungsan, Kyungbuk 712-714, Korea (e-mail: jwyun@taegu.ac.kr).

tion has been successfully established (Choi et al. 1999)

and these mushrooms will be able to be produced on a
large scale in the near future. Recently, many types of
polysaccharides from higher fungi have been reported to
have several physiological activities such as anticomplementary activity and antitumour activity (Chihara et al.
1970; Tabata et al. 1981; Song et al. 1998; Yang et al.
2000). Mass production of these strains subsequently
allows several Cordyceps species to be supplied for public
demands and employed as a target to search for a new
anticancer and immunomodulating drug (Kiho et al. 1996;
Kim et al. 2001).
Although many investigators have attempted to obtain
optimal submerged culture conditions for exo-polysaccharide
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EXO-POLYSACCHARIDE PRODUCTION BY CORDYCEPS MILITARIS C738

(EPS) production from several mushrooms, the nutritional

requirements and environmental conditions for submerged
cultures have not been extensively demonstrated (Yang and
Liau 1998; Park et al. 2001; Ricciardi et al. 2002). Moreover, submerged culture of C. militaris has scarcely been
studied even though it is viewed as a promising alternative
for effective production of its valuable metabolites, like
other mushrooms (Fang and Zhong 2002a; Nielsen et al.
1995). In the present study, the optimal culture conditions
for mycelial biomass and EPS production by C. militaris
C738 in submerged culture are investigated and the critical
effect of culture pH is described.

MATERIALS AND METHODS

Microorganism and media
A culture of Cordyceps militaris C738 was taken from our
culture collection, isolated from mountainous district in
Korea. The stock culture was maintained on potato dextrose
agar (PDA) slant. Slants were incubated at 25C for 7 d and
then stored at 4C. The seed culture was grown in a 250-ml
flask containing 50 ml of YM medium (10 g l)1 glucose,
5 g l)1 peptone, 3 g l)1 malt extract, and 3 g l)1 yeast
extract) at 25C on a rotary shaker incubator at 150
rev min)1 for 4 d.
Inoculum preparation and flask cultures
C. militaris C738 was initially grown on PDA medium in
a petri dish, and then transferred to the seed culture
medium by punching out 5 mm of the agar plate culture
with a sterilized self-designed cutter (Bae et al. 2000).
The seed culture was grown in a 250-ml flask containing
50 ml of basal medium at 25C on a rotary shaker
incubator at 150 rev min)1 for 4 d. The 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.

121

Analytical methods
Samples collected at various intervals from shake flask were
centrifuged at 10000 g for 20 min, and the resulting
supernatant was filtered by membrane filtration (045 lm,
2 Millipore Corporation, Bedford, MA, USA). The resulting
culture filtrate was mixed with four times its volume of
absolute ethanol, stirred vigorously and left overnight at
4C. The precipitated EPS was centrifuged at 10000 g for
10 min, discarding the supernatant. The precipitate of pure
EPS was lyophilized and the weight of the polymer was
estimated. Dry weight of mycelium was measured after
repeated washing of the mycelial pellet with distilled water
and drying overnight at 70C to a constant weight. The
filtrate from a membrane filtration was analysed by HPLC
(Shimadzu Co., Osaka, Japan) using an Aminex HPX-42C
column (078 30 cm, Bio-Rad Laboratories, Hercules,
3 CA, USA) equipped with a refractive index detector for
quantitative analysis of residual sugar concentration.
Rheological and morphological measurements
The rheological measurements were performed on samples
collected from the bioreactor at regular intervals using a
Brookfield programmable LVD-VII + digital viscometer
(Brookfield Engineering Laboratories Inc., Stoughton, MA,
4 USA) fitted with a small sample adapter. The morphological
properties of the samples collected were evaluated using an
image analyser (Matrox Electronic System Ltd, Dorval,
Quebec, Canada) with software coupled to a light microscope (Olympus Optical Co., Ltd, Tokyo, Japan) through a
CCD camera (Matsushita Communication Industrial Co.,
Ltd, Yokohama, Japan). The samples were fixed with an
equal volume of fixative (13 ml of 40% formaldehyde, 5 ml
glacial acetic acid with 200 ml of 50% ethanol). 01 ml of
each fixed sample was transferred to a slide, air dried, and
stained with methylene blue (03 g methylene blue, 30 ml
95% ethanol in 100 ml water) (Packer and Thomas 1990).
Estimation procedures

Bioreactor fermentations
The fermentation medium was inoculated with 4% (v v)
of the seed culture and then cultivated at 25C in a 5-l
stirred-tank fermenter (KF-250, KoBioTech, Seoul, Korea)
equipped with a pH electrode. Unless otherwise specified,
fermentations were conducted under the conditions of
temperature 25C, aeration rate 2 v v)1min)1, agitation
speed 150 rev min)1, initial pH 60, and working volume
3-l. The seed culture was transferred to the fermentation
medium and was cultivated for 4 d at 150 rev min)1
and 25C. All experiments were performed at least in
duplicate.

The pellet 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) (Riley et al. 2000). The specific
growth rate, l (h)1), was calculated from the equation:
l (1 X)(dX dt); where X is the cell concentration
(g l)1) at time t (h). The specific consumption rate of
substrate, QS X (g g)1 d)1) was estimated by the equation:
(dS dt)(1 X); where S is the concentration of sucrose

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122 S . - W . K I M ET AL.

(g l)1) at time t (d). The specific production rate of EPS,

PP X (g g)1 d)1) was estimated by the equation:
(dP dt)(1 X); where P is the concentration of EPS (g l)1)
at time t (d). The yield of EPS on substrate, YP S (g g)1)
was estimated by the equation: (dP dt) (dS dt).

Table 1 Effect of carbon and nitrogen sources on the mycelial

biomass and exo-polysaccharide production in Cordyceps militaris
C738*

RESULTS

Carbon source
Cellobiose
Dextrose
Fructose
Lactose
Maltose
Mannitol
Sorbitol
Sucrose
Xylose

Effect of Carbon and Nitrogen Source

To examine the effect of carbon sources on the production
of mycelial biomass and EPS, various carbon sources were
provided at 10 g l)1 instead of glucose as the carbon source
in the basal medium. As shown in Table 1, a high level of
mycelial biomass and EPS was obtained when cellobiose,
fructose, maltose or sucrose was used as the carbon source.
Of the carbon sources tested, the maximum EPS production
was obtained in the sucrose media of 60 g l)1 (Fig. 2a). The
effect of nitrogen source for the EPS production was studied
in the medium containing various nitrogen sources. As
shown in Table 1, the cells grown on the media containing
corn steep powder, meat peptone, polypeptone or yeast
extract produced a significantly high level of EPS. All
inorganic nitrogen sources gave rise to poor mycelial growth

Nitrogen source
Ammonium chloride
Ammonium citrate
Ammonium nitrate
Ammonium phosphate
Ammonium sulphate
Corn steep powder
Meat peptone
Polypeptone
Soypeptone
Yeast extract

Final
pH

887
894
813
587
800
867
867
859
662

046
037
033
052
043
045
033
065
038

436
467
518
698
531
610
545
495
463

242
396
356
448
322
2220
1670
1734
1160
2026

069
060
076
093
105
043
104
119
083
080

246
489
541
560
299
430
402
453
346
454

* Fermentations were carried out for 5 d at 25C.

The effect of initial pH (3090) on the kinetic aspects of

synthetic medium for fermentation of C. militaris C738 is
shown in Fig. 1(a). Maximum EPS concentration (029 g l)1)
was obtained in cultures grown at an initial pH 60. In
contrast, maximum biomass concentration (81 g l)1) was
obtained at an initial pH of 90. However, the difference
in EPS production according to the initial pH was not
so significant. To find the optimal temperature, this organism
was cultivated at various temperatures, where the optimum
temperatures for mycelial biomass and EPS production
were found to be 20C and 25C, respectively (Fig. 1b).

Exo-polysaccharide
(g l)1)

Effect of Initial pH and Temperature

Mycelial
biomass
(g l)1)

Fig. 2 Effect of sucrose (a) and polypeptone concentration (b) on the

mycelial biomass (d) and exo-polysaccharide production (s) in
C. militaris C738 in shake flask culture

and EPS production. Amongst organic nitrogen sources,

polypeptone yielded the best EPS production, where the
optimal concentration of polypeptone for EPS production
was 10 g l)1 (Fig. 2b).
Effect of Bioelements
Fig. 1 Effect of initial pH (a) and temperature (b) on the mycelial
biomass (d) and exo-polysaccharide production (s) in Cordyceps
militaris C738 in shake flask culture

The effect of different bioelements on mycelial growth and

EPS production by C. militaris C738 is shown in Table 2.

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EXO-POLYSACCHARIDE PRODUCTION BY CORDYCEPS MILITARIS C738

123

Table 2 Effect of bioelements on the mycelial biomass and exopolysaccharide production in Cordyceps militaris C738*

Bioelement

Mycelial biomass
(g l)1)

Exo-polysaccharide
(g l)1)

Final pH

Control
CaCl2
FeSO47H2O
KH2PO4
K2HPO4
MnCl26H2O

580
668
490
676
752
656

152
098
053
143
186
102

417
430
381
489
495
386

* Fermentations were carried out for 5 d at 28C.

 Control means no supplementation of bioelement.

The maximum production of EPS and biomass were found

in the medium containing K2HPO4. The significance of this
result is that large amounts of potassium ions are necessary
for efficient EPS production by C. militaris C738.
Fermentation Results
To investigate the effect of growth pH, which is one of
major factors for polysaccharide fermentation, two sets of
fermentations (pH-stat and without pH control) were
carried out in a 5-l stirred-tank fermenter. The typical time
courses of mycelial biomass and EPS were shown in Fig. 3.
In the case where pH was not controlled, initial pH declined
from 60 to 47 during exponential growth phase, followed
by return to the initial pH value towards the end of
fermentation (Fig. 3a). When the fermentation was carried
out with pH control (60), EPS production was significantly
increased (73 g l)1) in comparison with uncontrolled pH
condition (23 g l)1) (Fig. 3b). The overall growth kinetics
of C. militaris C738 with and without pH control is
comparatively illustrated in Table 3. Although the specific
growth rates of the cells were nearly the same irrespective of
pH control, the specific production rate and yield of EPS
were significantly higher at pH-stat culture. The fermentation results pointed out that pH-stat culture led to enhanced
production of EPS even though higher mycelial biomass was
achieved without pH control.

Table 3 Fermentation kinetics of Cordyceps

militaris C738 grown in the medium with
and without pH control

Fig. 3 The time profiles of mycelial dry weight and exo-polysaccharide production in a 5-l stirred-tank fermenter without (a) and with
pH control (b). Mycelial biomass (d); exo-polysaccharide production
(s); pH (n); residual sucrose (m)

Comparison of Morphological and Rheological

Properties
Figure 4 shows typical morphologies according to the
fermentation with and without pH control. The cells were
observed to form mainly pellets from the early stage and
were kept nearly constant during the entire period of fermentation. The morphological properties were characterized

Kinetic parameters

With pH
control

Without pH
control

Maximum biomass concentration, X (g l)1)

Maximum exo-polysaccharide concentration, P (g l)1)
Specific growth rate, l (h)1)
Specific consumption rate of substrate, Q S X (g g)1 h)1)
Specific production rate of exo-polysaccharide, PP X (g g)1 d)1)
Yield of exo-polysaccharide on substrate, YP S (g g)1)

1273
730
005
018
091
021

2570
230
006
013
010
003

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124 S . - W . K I M ET AL.

Fig. 4 The morpological change of Cordyceps militaris C738 in a 5-l stirred-tank fermenter without (A) and with (B) pH control

Fig. 5 The compactness and roughness of Cordyceps militaris C738

pellets growing in a 5-l stirred-tank fermenter without (d) and with
(s) pH control

by two parameters including roughness and compactness of

the pellets using an image analyser. Figure 5 shows the
roughness and compactness of the pellets during the entire
culture period with and without pH control. Both compactness and roughness of pellets decreased slowly with
sharp decrease of core area as the fermentations proceeded.
The rheological properties of culture broth were investigated to find out the effect of mycelial morphology on the
apparent viscosity of whole broth between the cultures with
and without pH control. Figure 6(a) shows the shear stress
vs shear rate for two fermentation broths. It can be clearly
seen that the rheological behaviour of the broth is obviously
pseudoplastic irrespective of culture condition. The apparent viscosities of the whole broth according to the fermentation period for two different fermentations are depicted in
Fig. 6(b). The viscosities of both fermentation broth
increased rapidly after 3 d of the fermentation. The highest
EPS production was achieved at day 10 when the apparent
viscosity indicated the highest value (Fig. 3). It can be

Fig. 6 Shear rate vs shear stress (a) and the apparent viscosity (b) of
the fermentation broth Cordyceps militaris C738 in a 5-l stirred-tank
fermenter without (d) and with pH control (s)

clearly seen that the apparent viscosity was much influenced

by EPS concentration than that by mycelial biomass.
DISCUSSION
The nutritional requirement for EPS production in basidiomycetes and ascomycetes differs in strains and culture

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EXO-POLYSACCHARIDE PRODUCTION BY CORDYCEPS MILITARIS C738

conditions. Moreover, different carbon source can result in

the different carbohydrate compositions in polysaccharides.
The optimal carbon and nitrogen source for the mycelial
growth and EPS production in C. militaris C738 were in
accordance with those obtained by other investigators (Bae
et al. 2000; Kim et al. 2002; Park et al. 2002). Maltose is also
known as an efficient carbon source for EPS production in
liquid-cultures of mushrooms (Bae et al. 2001; Kim et al.
2002). Considering the cost and feasibility in handling,
maltose and sucrose are regarded as more efficient substrate
ingredients than other compounds such as soluble starch and
sugar alcohols.
It has been known that several complex nitrogen sources
such as polypeptone and corn steep powder were desirable
while inorganic nitrogen sources were inefficient for the
production of EPS in submerged cultures of mushrooms
(Bae et al. 2001; Kim et al. 2002). Several amino acids were
also proved to be a more favourable nitrogen sources than
complex nitrogen sources for mycelial growth of an
antitumour mushroom, Phellinus linteus (Chi et al. 1996).
Many investigators claimed that the different morphology
of fungal mycelia under a different initial pH value was the
critical factor in biomass accumulation and metabolite
formation (Wang and McNeil 1995; Lee et al. 1999; Fang
and Zhong 2002b). The medium pH may affect cell
membrane function, cell morphology and structure, the
solubility of salts, the ionic state of substrates, the uptake of
various nutrients, and product biosynthesis. In general, cells
can only grow within a certain pH range, and metabolite
formation is also often affected by pH. For example, EPS
production in Sclerotium glucanicum was greatly affected by
culture pH (Wang and McNeil 1995). Fang and Zhong
(2002b) reported that lowering the initial pH from 65 to 35
gradually led to higher production of EPS and a higher
specific production of intracellular polysaccharide in a
popular basidiomycete, Ganoderma lucidum. Lee et al.
(1999) suggested that the bistage pH control technique
was applied for enhanced EPS production from in an airlift
fermenter, by which one pH value for optimal mycelial
growth was shifted to another value in the liquid-culture of
Ganoderma lucidum.
Detailed quantitative structural information of mycelial
clump and pellet morphologies is required for a better
understanding of the relationship between morphology and
target metabolite production. In this respect, the capabilities of image analysis are essential to obtain morphological
parameters. However, there are conflicting reports on the
relationship between fungal morphology and productivity
of objective metabolites. Sinha et al. (2001a, b) suggested
that the pellets with high compactness are the more
productive morphological form compared to free filamentous mycelia in Paecilomyces japonica fermentation. In
contrast, Park et al. (1999) demonstrated that arachidonic

125

acid production was preferred when Mortierella alpina cell

5,6 exhibited a feather-like mycelial clump of small size.
Paul et al. (1999) reported that pelleted fermentation of
Aspergillus niger gave low citric acid production but also
high by-product impurities. Taking into account that
higher polysaccharides were produced under pH-stat
culture in this work, larger and more compact pellets were
desirable for EPS production in C. militaris. Similar
observations have been reported by other investigators for
several kinds of ascomycetes (Park et al. 2001; Park et al.
2002). Putting the results from different investigators
together, the desirable morphology for the production of
each objective metabolite indeed differs in nature of
organisms.
Fermentation broth rheology greatly affects transport
processes in the bioreactor which, in turn, have a strong
influence on the efficiency and productivity of the entire
fermentation process. Mycelial fermentation broths are
initially Newtonian because of low biomass concentrations.
However, with time, there is an increase in biomass
concentration, which considerably alters the rheology of
the fermentation broth to usually pseudo-plastic behaviour
(Roels et al. 1974). Relatively few studies have been aimed at
quantifying the influence of biomass concentration on the
broth rheology. Penicillium chrysogenum is a good example in
that the viscosity is higher when the cells are growing as
filament instead of as pellets (Warren et al. 1995). It has
been shown that correlations can be found between broth
rheological parameters and fungal morphology (Olsvik et al.
1993; Tucker and Thomas 1993). The apparent viscosity of
the whole broth from C. militaris C738 fermentation gave
7 non-significant high values (100170 cP) due to the low
viscous nature of EPS produced, which, surprisingly, is
lower than those from other mushroom fermentations
(Sinha et al. 2001a,b; Cho et al. 2002) (Fig. 6b). This
rheological feature is very advantageous for large-scale
production of EPS.
ACKNOWLEDGEMENT
This work was supported by Taegu University Research
Grant, 2002.
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