effects of cultivating conditions on the mycelial growth

Effects of cultivating conditions on the mycelial growth
of Ganoderma lucidum in submerged flask cultures
F C. Yang, C B. Liau
Abstract In this paper the effects of environmental con-
ditions on the mycelial growth of Ganoderma lucidum
were investigated in shake ¯ask cultures. The optimal
temperature and pH were found to be around 30±35 °C
and 4, respectively, in a glucose-ammonium chloride me-
dium. The maximum mycelial concentration reached to
around 350 mg/100 ml. The formation of mycelial pellets
and their ultra structure was demonstrated to be greatly
affected by cultivating conditions. Increasing surface ae-
ration would be bene®cial for mycelial growth. However,
too high rotating speed in shake cultures had a detrimental
effect on the formation of mycelial pellets and the opti-
mum was found to be 100 rpm.
1
Introduction
Ganoderma lucidum (Fr.) Karst (Polyporaceae) is a species
of basidiomycetes which belongs to polyporacceae (or
Ganodermaceae) of Aphyllophorales. Its fruiting body is
called ``Reishi'' in Japanese and ``Lingzhi'' in China. In the
regions of China, Japan and Korea, Lingzhi has been a
popular folk or oriental medicine to cure various human

diseases, such as hepatitis, hypertension, hype-
rcholesterlemia and gastric cancer. Recent studies on this
fungus have demonstrated many interesting biological
activities, including antitumour, and anti-in¯ammatory
effects and cytotoxicity to hepatoma cells. These studies
also suggested that the carcinostatic substance in Lingzhi
is a polysaccharide, b-(1- > 3) -D-glucan [1, 2]. This
polysaccharide seems to show promise as a new type of
carcinostatic agent which might be useful in immuno-
therapy. Lingzhi, because of its perceived health bene®ts,
has gained wide popularity as a health food, in both Japan
and China. Reishi cultivation has prospered in Japan,
China, Taiwan, and Korea. In addition, attempts are being
made to obtain useful cellular materials or to produce
effective substances from cultured mycelia [1±3].
Mushroom mycelia or spawns have normally been
produced in solid cultures using substrates such as grain,
sawdust or wood. Propagation of edible mushrooms in
submerged culture was initially developed during the
1950s based on the success of growing lower fungi in
fermenters for economical production of various natural
products. Since then numerous attempts have been made
by researchers to cultivate mushroom mycelium com-
mercially in submerged culture[4]. Submerged culture has
the potential advantage in that it can be dispersed within
the substrate more uniformly than solid spawn and the
time taken to produce the ®rst crop of sporophores may be
shortened. Further, the liquid nature of such spawn en-
ables inoculation to be carried out under relatively more
stringent aseptic conditions which is important when

using non-selective substrates[4±6].
Yields and productivity of mushroom mycelium vary
widely, depending on the mushroom, substrate, and con-
ditions. Although many workers have attempted to obtain
mycelium of Ganoderma lucidum using submerged cul-
ture, very little information is available regarding the
environmental factors affecting mycelial growth of
G. lucidum in submerged culture[2, 3]. The study reported
here was carried out to determine the physical conditions
required for the mycelial growth of G. lucidum in sub-
merged shake cultures. In this paper, we also report factors
affecting the formation of mycelial pellets and their ultra
structure[7].
2
Materials and methods
2.1
Microorganism and media
The culture used was Ganoderma lucidum CCRC 36123
obtained from the Culture Collection and Research Centre
(CCRC), Food Industry Research and Development Insti-
tute (Hsinchu, Taiwan). Culture was maintained on potato-
dextrose-agear slope. Slopes were inoculated and incubated
at 30 °C for 7 days, and stored at 4 °C. The media were made
up of the following components (in gram per liter): glucose
50; K
2
HPO
4
0.5, KH
2

PO
4
0.5, MgSO
4
á 7H
2
O 0.5 yeast
extract 1 and ammonium chloride 4.
2.2
Cultivation of microorganism
The shake-¯ask experiments were performed in 500-ml
Erlenmeyer ¯asks containing 100 ml of the media. Media
were sterilized at 120 °C for 20 min and glucose was au-
toclaved separately. The pH was measured and adjusted to
Bioprocess Engineering 19 (1998) 233±236 Ó Springer-Verlag 1998
233
Received: 21 October 1997
F C. Yang, C B. Liau
Department of Chemical Engineering, Tunghai University,
Taichung, Taiwan 40704, R.O.C
Correspondence to: F C. Yang
The authors wish to thank the National Science Council of R.O.C.
for ®nancial supports (NSC 85-2214-E-029-004).

the desired value by addition of either 4M-HCl or 2.5M
NaOH. Actively growing mycelia (4 pieces, each
5mm´ 5 mm) from a newly prepared slant culture
(about 7 days incubation at 30 °C) were inoculated into the
¯ask. The ¯asks were shaken on a New Brunswick rotary
shaker (Model G24) at 100 rpm and 30 °C.
At the end of inoculation period mycelium consisting of
individual pellets was harvested by centrifugation
and wash for the analysis. The yield was expressed as
mg/100 ml dry weight. The range in size of individual
mycelium pellet was determined by measuring the average
diameter of pellets per culture.
2.3
Analytical methods
The pH was measured with a digital pH meter (Suntex,
Taiwan, model 2000A). Due to the fact that mycelia and
cell-bound polysaccharide could not be thoroughly sepa-
rated by centrifugation, in order to determine the con-
centrations of mycelium and polysaccharide, samples were
®rst subjected to ultrasonication for 2 hrs in a Branson
ultrosonicator (model 5210). Centrifugation was then
performed to remove cells and cell debris in a centrifuge
(Hettich, model ERA3S/10 ml). Dry weights of total cell
mass were obtained by centrifuging samples at 3000 rpm
for 10 min, washing the sediment three times with water,
and drying to constant weight.
3
Results and discussion
3.1
Effect of initial pH

The mycelia of various species of mushrooms will grow
over a wide range of pH values. However, for most or-
ganisms, the most favorable pH range is from 5 to 7. The
optimal initial pH for growth was determined for the se-
lected strain in glucose-ammonium chloride medium or
glucose-malt extract medium over a pH range of 3.0 to 6.0,
incubating for 7 days or 14 days. The optimum pH for the
highest yield of G. lucidum in a glucose-ammonium
chloride medium was 4.0 as shown in Fig. 1. However, it is
interesting to note that the optimal pH for G. lucidum
growing in a glucose-malt extract medium was found to be
around 5.0. It demonstrated that optimal initial pH for
mycelial growth would depend on the culture medium.
Lower values of initial pH would be bene®cial to inhibit
the growth of bacterial contaminants. In general, when
ammonium salts were used as the nitrogen source, the pH
decreased during the mycelium growth as the result of
assimilation of the ammonium ion and the attendant
effects of acids anions in the medium such as chlorides,
sulfate, or phosphate. The lower limit of this pH decrease
depends upon the buffering action of the constituents of
the medium and the mycelium[4].
3.2
Effect of cultivating temperature
A narrow temperature range for submerged culture of
G. lucidum mycelia has been reported. The results in Figs.
2 and 3 show that the optimal growth temperature of
Fig. 1. Effect of initial pH on the growth of mycelium of
G. lucidum in the glucose-NH
4

Cl medium on a rotary incubator
at 100 rpm and 30 °C
Fig. 2. Effect of cultivating temperature on the growth of myce-
lium of G. lucidum in the glucose-NH
4
Cl medium on a rotary
incubator at 100 rpm and initial pH 4.0
Fig. 3. Effect of cultivating temperature on the growth of myce-
lium of G. lucidum in the glucose-malt extract medium on a
rotary incubator at 100 rpm and initial pH 5.0
234
Bioprocess Engineering 19 (1998)







G. lucidum in glucose-malt extract medium or glucose-
ammonium chloride medium was found to be 30±35 °C.
The growth rate of this organism decreases rapidly above
and below these values. The lower values of ®nal pH seem
to indicate better growth of G. lucidum mycelia.
3.3
Effect of surface aeration
The effects of the surface aeration were investigated by
varying the volume of ¯ask so that the ratio of the surface
area of the liquid exposed to air to the liquid volume was
varied, affecting the aeration during the shaking process.

Different volumes of Erlenmeyer ¯asks (250 ml and
500 ml) with or without baf¯e were ®lled with the medium
of 100 ml and shaken in a standard manner. Table 1 shows
that increased surface aeration enhanced the ®nal dry
mycelium concentration obtained in the fermentations
and the best yield could be achieved when a baf¯ed 500 ml
Erlenmeyer ¯ask was used in a 7 days fermentation.
However, the mycelium form varied. Larger pellets were
produced at lower surface aeration rates, corresponding to
a lower dry mycelium concentration. Smaller pellets were
obtained when surface aeration rates increased, corre-
sponding to a higher dry mycelium concentration.
The formation of pellets is largely determined by the
extent of agitation and aeration. The effect of aeration on
the growth of mushroom mycelium, speci®cally in the
development of a pelleted mycelium, is complex and, in
some cases, contradictory. In general pellet formation is
favoured by low agitation and aeration rates. The oxygen
mass transfer in the fermentation suspension is enhanced
when pellets are formed because of the lower resistance
produced by lowering the viscosity of the medium, as
compared with ®lamentous growth in the same type of
medium. However, the oxygen supply to the interior of the
pellets decreases because of the condensation of mycelium
characteristics of the pellet structure and as a function of
the pellet diameter. Diffusion of oxygen from the medium
into large pellets is assumed to be the limiting factor for
growth of mushroom mycelium.
According to the paper of Litch®eld in 1967[4], aeration
rates in the range used by other common aerobic fer-

mentations are usually detrimental to mushroom myceli-
um growth. In some cases of cultivating mushrooms in
submerged culture, it was observed by several investiga-
tors that increasing aeration rate resulted in ®lamentous
growth, and reduced yield. The effect of high aeration rate
on the growth of mycelium of G. lucidum should be
studied further by using the fermenter.
3.4
Effect of shaking frequency
The in¯uence of rotating speed on mycelium growth was
studied in the range of 50±250 rpm while all other
conditions were kept constant. As to the results shown in
Fig. 4, the maximum concentration of mycelium was ob-
served at the shaking frequency of 100 rpm. There was an
increase in the yield of mycelium when the shaking fre-
quency was increased from 50 to 100 rpm. It is supposed
that a higher rpm implies a better oxygen transfer in the
fermenting medium. However, the fact that the biomass
yields were lower above 100 rpm could be attributed to a
detrimental effect of increased shear stress on the myce-
lium.
The sizes of the pellets formed and their distributions
were mainly affected by the rotating speed. As to the re-
sults shown in Fig. 5, the size of mycelial pellets decreased
with increase in shaking frequency. At low rotating speed,
larger mycelial pellets were formed and the pellets formed
were very incompact in nature and were loosely arranged.
However, at higher speed, due to the excessive shear force,
the pellets formed were extremely tiny in size. This may
Table 1. Effects of surface aeration on the growth and ultra structure of mycelium in ¯ask cultures of G. lucidum

Flask
No.
Mycelium conc.
(mg/100 ml)
Final pH Pellet number Pellet diameter
(mm)
(1) 144 5.05 21 10
(2) 232 4.64 9 14
(3) 479 3.97 100 3
(4) 555 3.88 150 2
1. Flask No. (1). The medium of 100 ml in 250 ml-Erlenmeyer ¯asks without baf¯e
(2). The medium of 100 ml in 250 ml-Erlenmeyer ¯asks with baf¯e
(3). The medium of 100 ml in 500 ml-Erlenmeyer ¯asks without baf¯e
(4). The medium of 100 ml in 500 ml-Erlenmeyer ¯asks with baf¯e
2. under the conditions of initial pH = 5.6, 30 °C and 100 rpm for 7 days
3. malt extract 4 %, yeast extract 0.1 %, K
2
HPO
4
0.05 %, KH
2
PO
4
0.05 %, MgSO
4
á 7H
2
O 0.05 %
Fig. 4. Effect of rotating speed on the growth of mycelial of
G. lucidum in the glucose-NH

4
Cl medium on a rotary incubator
at 100 rpm and 30 °C
235







indicate that G. lucidum is an obligate aerobe and the
optimum rotating speed is around 100 rpm.
3.5
Effect of size of inoculum
In order to enhance cell density and also to develop a
method for large scale cultures, various sizes and types
of inoculum were tested. Inoculum was prepared by
submerged culture using a shake ¯ask for 7 days. After
agitation in a blender for 5 seconds, the mycelia were
inoculated into the ¯asks with 4 levels of inoculum (1, 4, 8,
12 ml per 100 ml broth). After 7-day cultures at 30 °C and
100 rpm, the ®nal mycelia concentrations were 235, 351,
462, and 579 mg per 100 ml, respectively. In contrast to
inoculation from a slant, when submerged culture was
used for inoculum, the mycelial pellets became smaller and
more uniform in size. The results showed that an increase
in inoculum concentration increased the yield of myceli-
um and the number of pellet but decreased the size of
mycelial pellets. However, when too many pellets were

present in the broth, some tiny particles could stick to-
gether during the culture and caused the increase of pellet
size.
4
Conclusions
As described above, yields and productivity of mushroom
mycelium vary widely, depending on the mushroom,
substrate, and conditions. Effects of some environmental
factors on the growth of mycelia of G. lucidum were
investigated in shake ¯ask culture in this report. The
formation of mycelial pellets and their ultra structure was
also demonstrated to be greatly affected by cultivating
conditions. However, in order to meet the requirement
of large scale production, further study about the effect
of agitation and aeration on the growth of mycelia of
G. lucidum in fermenter cultures would be necessary.
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Fig. 5. Effect of rotating speed on the ultrastructure of mycelial
pellet of G. lucidum in the glucose-NH
4
Cl medium on a rotary
incubator at 100 rpm and 30 °C
236
Bioprocess Engineering 19 (1998)