Enzyme and Microbial Technology 27 (2000) 295–301 www.elsevier.

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Effect of fatty acids on the mycelial growth and polysaccharide

formation by Ganoderma lucidum in shake flask cultures夞
Fan-Chiang Yang,* Yn-Fuu Ke, Shanq-Shin Kuo
Department of Chemical Engineering, Tunghai University, Taichung, Taiwan 40704, R.O.C.
Received 4 November 1999; received in revised form 9 February 2000; accepted 15 March 2000

Abstract

Fatty acids were added into the media to investigate their effects on the mycelial growth and polysaccharide formation by Ganoderma
lucidum. The experiments were carried out in freely suspended cultures or immobilized cultures using shake flasks. The results indicate that
the extent of stimulation or inhibition were associated with the types and levels of fatty acids. Oleic acid at the level of 0.15 g/100 ml led
to a significant increase in cell concentration from 0.20 to 0.46 g/100 ml in a suspended culture and palmitic acid was of great advantage
to polysaccharide production. In contrast, linoleic acid (0.1 g/100 ml) drastically suppressed both mycelial growth and polysaccharide
formation. In immobilized cultures with fatty acids, the stimulation of mycelial growth remained the same level, but the enhancement of
polysaccharide production became less. In addition, the growth of G. lucidum in the pattern of immobilization might be beneficial to the
production of mycelia and polysaccharide. © 2000 Elsevier Science Inc. All rights reserved.

Keywords: G. lucidum; Polysaccharide; Mycelial growth; Fatty acids

1. Introduction cause it normally takes 6 months to complete a fruiting body

Ganoderma lucidum (Fr.) Karst (Polyporaceae) is a spe-
cies of basidiomycetes that belongs to polyporacceae (or
Ganodermaceae) of Aphyllophorales. Its fruiting body is
called “Reishi” in Japanese and “Lingzhi” in China. For
centuries, this mushroom has been regarded in the Orient as
a popular folk or effective medicine used to treat various
human diseases, such as hepatitis, hypertension, hypercho-
lesterolemia, and gastric cancer. Recent studies on this fun-
gus have demonstrated that the carcinostatic substance in
Lingzhi is a polysaccharide, 1, 3-␤-D-glucan. This polysac-
charide seems to have promise as a new type of carcino-
static agent, which might be useful in immunotherapy [1–3].
Because of its perceived health benefits, Lingzhi has
gained wide popularity as a health food and the cultivation
has prospered in the regions of China, Japan, Korea, and
Taiwan, which have normally been produced in solid cul-
tures using substrates such as grain, sawdust or wood. Be-
夞 The authors wish to thank the National Science Council of R.O.C. for
financial supports (NSC 87-2214-E-029-003).
* Corresponding author. Tel.: ⫹886-43590262 ext. 214; fax: ⫹886-
43590009
E-mail address: fcyang@mail.thu.edu.tw (Fan-Chiang Yang).
culture in solid state fermentation, many attempts are being
made to obtain useful cellular materials or to produce ef-
fective substances from a submerged mycelia culture [2,3].
In addition, the mycelia of G. lucidum have been cultured in
the pattern of immobilization to investigate the feasibility of
continuous operation for the production of polysaccharide
in our laboratory [4]. Apart from exopolysaccharide, some
components extracted from liquid-cultured mycelia were
also demonstrated to be bioactive [1]. The process of a
submerged culture for mycelial growth of G. lucidum has
been very well developed in Taiwan in recent years, in
which mycelial biomass and polysaccharide are the desired
products [5,6].
To accelerate mycelial growth of some mushroom spe-
cies, plant oils have been proved to have a stimulatory effect
[7]. In addition, many workers have also reported that fatty
acids, oils and surfactants promoted the production of fun-
gal metabolites like citric acid, aflatoxins, and carotenes as
well as exocellular enzymes [8 –10]. It was also reported
that the production of microbial exopolysaccharide was
stimulated with some fatty acids [11]. Their effects may be
due to depression, induction or stimulation of secretion. In
our preliminary experiments, vegetable oils tested at certain
levels did stimulate mycelial growth and polysaccharide
formation by G. lucidum; however, higher concentrations

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PII: S 0 1 4 1 - 0 2 2 9 ( 0 0 ) 0 0 2 1 3 - 1
296 F.-C. Yang et al. / Enzyme and Microbial Technology 27 (2000) 295–301
might also cause an inhibitory effect [4]. The expression of
enhancement or inhibition might be dependent upon the
types of fatty acids present in oil. This research describes
experiments carried out to investigate the effects of fatty
acids on mycelial growth and polysaccharide formation by
G. lucidum in freely suspended cultures or immobilized
cultures.
2. Materials and methods
2.1. Organism and inoculum
G. lucidum CCRC 36123 was obtained from the Culture
Collection and Research Center (CCRC), Food Industry
Research and Development Institute (Hsinchu, Taiwan).
Cultures were maintained on potato-agar-dextrose slopes.
Slopes were inoculated and incubated at 30°C for 7 days,
and stored at 4°C. To prepare the inoculum, the mycelium
of G. lucidum was transferred to petri-dish containing PDA
medium at 30°C for 7 days. Mycelial agar discs (0.5 cm)
were obtained by a self-designed cutter and used as the
inoculum in a shake flask culture [5,6].
2.2. Suspended or immobilized cultures using shake flasks
A shake flask culture was incubated in a 250 ml Erlen-
meyer flask with a silicone plug, which contained 100 ml of
the medium. The media were made up of the following
components (in grams per liter): glucose, 50; K2HPO4 0.5;
KH2PO4 0.5; MgSO4 7H2O 0.5; yeast extract 1; and am-
monium chloride 4. The pH was adjusted to the desired
value by addition of either 0.1 N HCl or 2.5 M NaOH. For
an immobilization culture, a polyurethane foam sheet was
used as a carrier, which was purchased from Chyan–Fwu
Co., Taichung, Taiwan. The foam sheet with the dimensions
of 4 ⫻ 4 ⫻ 2 cm and the porosity of 0.98 was weighted and
placed in the flasks before sterilization. Media were steril-
ized at 120°C for 20 min and glucose was autoclaved
separately. The flasks were incubated on a New Brunswick
rotary shaker (Model G24) under the conditions of 100
rev./min and 30°C for 7 days. Fatty acids were purchased
from Sigma Biochemicals (St. Louis, MO, USA).
2.3. Sampling
Due to the fact that the pellets were formed during the
suspended culture of mycelium, taking a sample from a
flask by a pipette was difficult or even impossible. There-
fore, whether the mycelia were grown in free suspension or
in immobilization, one flask was required for each assay and
a fermented broth of 100 ml containing biomass was used
for the determination of biomass and polysaccharide. Two
sets of shake flasks were prepared at the same time for each
test. The values are means of duplicate determination.
2.4. Biomass concentration
The concentration of fungal biomass in freely suspended
cultures was determined by filtering the cells through pre-
weighted filterpaper (GF/C Whatman) under suction, wash-
ing the filter three times with water, and drying to constant
weight at 80°C. All filtrates were collected for the determi-
nation of polysaccharide. To monitor the growth of immo-
bilized cell cultures, the foam sheet with the dimensions of
4 ⫻ 4 ⫻ 1 cm was weighted and placed in the flasks in the
immobilization test. The foam matrices filled with immobi-
lized cells were carefully drained, washed, and dried to
constant weight at 80°C.
2.5. Polysaccharide concentration
All filtrates were collected, and then the crude polysac-
charide was precipitated with the addition of four volumes
of 95% ethanol. The precipitated polysaccharide was col-
lected by centrifugation at 3000 rev./min for 10 min and
then dried to remove residual ethanol at 60°C. Total poly-
saccharide in the culture medium was determined by phe-
nol-sulphuric acid assay according to Dubois et al. [12,13].
The pH was measured with a digital pH meter (Suntex,
Taiwan, model 2000A).
3. Results and discussion
3.1. Effect of addition of plant oils on the production of
mycelia and polysaccharide
The effects of plant oils on the production of mycelia and
polysaccharide by G. lucidum were studied in freely sus-
pended cultures. The results in Table 1 reveal that all plant
oils at the level of 1% were beneficial to cell growth and led
to the increase of biomass concentrations. Olive oil was the
most effective and cell concentration rose from 0.20 to 0.33
g/100 ml. Concerning the effect on polysaccharide forma-
tion, the increase with safflower oil was marked and the
presence of other oils did not result in a significant change.
However, soybean oil decreased the production slightly.
The yield of polysaccharide from biomass presented as mg
polysaccharide/mg biomass is listed in the third column of
the table, and varied from 0.044 to 0.065. Apparently, the
values indicate that there was no significant relation be-
tween the yield and the addition of plant oils, which is
consistent with the results of the reference paper [10]. This
could be partially attributed to the fact that the increases of
biomass and polysaccharide occur simultaneously. As de-
scribed above, since biomass and polysaccharide are the
desired products, the increase of total production amount or
production rate by the addition of plant oils or fatty acids
would be beneficial to the process. According to our pre-
liminary tests, higher levels caused an inhibitory effect [4].
The expression of enhancement or suppression might be
 F.-C. Yang et al. / Enzyme and Microbial Technology 27 (2000) 295–301
Fig. 1. Production of biomass and polysaccharide by G. lucidum with time
in suspended cultures in the presence and absence of palmitic acid (0.1
g/100 ml) at 30°C, initial pH 4 and 100 rpm.
determined by the types of fatty acids present in oil and
were examined in the following tests.
3.2. Production of biomass and polysaccharide with time
Time courses of concentrations of biomass and polysac-
charide produced in suspended culture of G. lucidum are
Fig. 2. Effect of free fatty acids on the mycelial growth of G. lucidum in freely suspended cultures at 30°C, initial pH 4 and 100 rpm for 7 days.
297
shown in Fig. 1. These results indicate that the production of
polysaccharide increased in parallel with the growth of
mycelium and the pattern of product is the type of growth
associated. The addition of palmitic acid at the level of 0.1
g/100 ml stimulated both polysaccharide and biomass pro-
duction at an increased rate. Compared to the control, the
lag phase was shortened substantially from 4 to 2 days and
the biomass concentration increased from 0.23 to 0.42 g/100
ml on the 8th day. Polysaccharide concentration also rose
more than 40%. On the basis of 7-days cultures, the effects
of different type and level of fatty acids are compared as
follows.
3.3. Effect of addition of fatty acids on the mycelial
growth
Fig. 2 shows the effect of various types and levels of
fatty acids on mycelial growth for 7 days. Palmitic acid,
oleic acid, and stearic acid showed a stimulatory effect.
Oleic acid was the most impressive and with the optimum of
0.15 g/100 ml, mycelial concentration increased signifi-
cantly from 0.20 to 0.46 g/100 ml. In contrast, it is surprised
to note that the addition of high level of linoleic acid (0.1
g/100 ml) drastically suppressed mycelial growth and no
biomass was observed. These data also indicate the lack of
any correlation between growth stimulation and the extent
of unsaturation of fatty acids. After a preliminary test no cell
was grown in an immobilized culture in the presence of
linoleic acid. To understand the effect of fatty acid with
298 F.-C. Yang et al. / Enzyme and Microbial Technology 27 (2000) 295–301

Fig. 3. Effect of free fatty acids on the mycelial growth of G. lucidum in immobilized cultures at 30°C, initial pH 4 and 100 rpm for 7 days.

shorter chain, linoleic acid was then replaced by capric acid
in immobilized cultures. The results of mycelial growth
with fatty acids in immobilized cultures are given in Fig. 3
and these are basically in accordance with the data de-
scribed above. Oleic acid was still the stimulatoriest and at
the level of 0.1 g/100 ml cell concentration had a 2-fold
increase. The presence of palimitic acid and stearic acid was
also advantageous. However, capric acid (a shorter-chain
fatty acid C 10) inhibited the growth. This is consistent with
the results of Fukushima et al. (1991) [8]. Accordingly,
growth of Aspergillus oryzae was entirely inhibited by ca-
pric acid, caproic acid, propionic acid, and acetic acid, all
with carbon chain lengths shorter than 10.
Compared with the results listed in Table 1, the stimu-
latory effect from olive oil might be attributed to the fact
that the main composition of the oil is oleic acid [14].
However, the inhibition caused by linoleic acid was incon-
sistent with the effect of safflower oil, in which the main
type of fatty acid is linoleic acid [15]. The elimination of the
suppression might be attributed to other types of fatty acids
present in safflower oil. The mechanism of stimulation has
been suggested as the lipids may be partially incorporated in
the cell membrane thereby facilitating immediate uptake of
nutrients from the culture medium. According to the paper
[16], the major fatty acid in some fungus was an di-unsat-
urated 18 carbon acid. The effect of linoleic acid (C18) on
mycelial growth and lipid composition of mycelium of G.
lucidum would be of interest and deserve further study.

Table 1
Effect of adding plant oils on the mycelial cultures of G. lucidum

Plant oil Mycelium conc. Polysaccharide conc. Yield (mg Final pH

(mg/100 ml) (mg/100 ml) polysaccharide/
mg biomass)

Control 202.1 12.6 0.062 2.91

Soy 232.3 10.1 0.044 3.27
Peanut 256.0 16.2 0.063 3.34
Safflower 277.7 18.2 0.066 3.14
Corn 306.2 15.5 0.051 3.19
Sunflower 293.9 15.7 0.053 3.10
Olive 328.3 15.9 0.048 2.83

The suspended cultures were performed in 250 ml flasks containing 1% plant oils at 30°C, initial pH 4 and 100 rev./min for 7 days.
 F.-C. Yang et al. / Enzyme and Microbial Technology 27 (2000) 295–301
Fig. 4. Effect of free fatty acids on polysaccharide production by G. lucidum in freely suspended cultures at 30°C, initial pH 4 and 100 rpm for 7 days.
3.4. Effect of addition of fatty acids on the polysaccharide
production
When a selection of individual fatty acids was tested, the
results of polysaccharide production in suspended cultures
are given in Fig. 4. They show that palmitic acid and oleic
acid, at the levels tested (below 0.25 g/100 ml), had a
positive effect on the formation of polysaccharide, which
increased proportionally with the amount of fatty acids
added. However, stearic acid showed the stimulation only at
the lower concentration and the optimum at the level of 0.1
g/100 ml. In contrast, linoleic acid had a strong inhibitory
effect on the polysaccharide formation and this is in agree-
ment with the results of Stasinopoulos and Seviour in 1990,
in which exopolysaccharide production by Acremonium
persicinum was inhibited with fatty acid of linoleic acid
[11]. Based on the results of this study, the correlation
between stimulatory effect and the extent of unsaturation of
fatty acids tested could not be determined.
The effect of addition of fatty acids on the polysaccha-
ride formation in immobilized cultures is shown in Fig. 5.
Compared with the data in Fig. 4, fatty acids became less
stimulatory. Oleic acid and stearic acid at the levels tested
had little effect on polysaccharide production and only
palmitic acid was stimulatory. Capric acid (C 10) sup-
pressed the production.
Results presented here indicate that the synthesis of ex-
opolysaccharide by G. lucidum can be substantially in-
creased by the presence of certain plant oils and fatty acids
299
in the medium. Although these compounds have been re-
ported to stimulate production of other fungal metabolites,
their effect on polysaccharide formation has only been ex-
amined in few studies. The mechanism of stimulatory effect
has been proposed as oils or fatty acids work by modifying
membrane composition and increase permeability, or by
directly affecting the level of synthesis of the enzymes
involved in polysaccharide production [11]. In this study,
the phenomena of stimulating both mycelial growth and
polysaccharide formation might suggest that their role be
probably explained in terms of membrane structure and
function.
3.5. Comparison of freely suspended cultures and
immobilized cultures
The comparison of freely suspended and immobilized
cultures of G. lucidum without oil addition can be made
from the controls in Figs. 2–3 and 4 –5. Without foam
supporting material in the flask the mycelium aggregated
into small beads with the diameter of around 5-mm. The
sizes of the pellets formed and their distributions were
mainly affected by the rotating speed [5]. If the foam matrix
(4 ⫻ 4 ⫻ 2 cm) was employed, the mycelia all adhered to
the surface of foam particle and there were almost no freely
suspended mycelia in the bulk liquid. It is of interest to note
that the growth of G. lucidum in the pattern of immobiliza-
tion was beneficial to the production of mycelial growth and
polysaccharide. The supply of solid materials might be
300 F.-C. Yang et al. / Enzyme and Microbial Technology 27 (2000) 295–301
Fig. 5. Effect of free fatty acids on polysaccharide production by G. lucidum in immobilized cultures at 30°C, initial pH 4 and 100 rpm for 7 days.
advantageous for cell attachment and mycelial growth, and
this led to the increase of polysaccharide formation. How-
ever, the carrier definitely made the polysaccharide secre-
tion slow. As described above, due to the fact that it takes a
long period of time to grow, the immobilized mycelia of G.
lucidum have been used to investigate the feasibility of
continuous operation for the production of polysaccharide
in another project in our laboratory [4]. Accordingly, when
the culture period was prolonged to 2–3 weeks, a large
portion of polysaccharide produced tended to adhere to the
foam matrix. Although the idea of continuous operation
proved to be immature, immobilization may provide an
alternative way for the recovery of product (biomass and poly-
saccharide) and further study is required for the evaluation.
4. Conclusions
The production of mycelia and polysaccharide by some
mushroom species in submerged cultures has prospered in
the Orient in recent years. In order to enhance the produc-
tion efficiency, the control of environmental conditions or
the modification of media composition would be vital [4,5].
According to the reference paper [10], in order to control
foam formation accompanying ␤-glucan production by A.
persicinum, some of plant oils used as antifoams were
unexpectedly found to increase glucan production. Thus,
when an antifoaming agent has to be employed in a large-
scale liquid culture of G. lucidum, some plant oils deserve to
be considered to accelerate the production process, although
the type and the level has to be determined with caution.
The addition of oils or fatty acids was demonstrated to have
a great influence on the production of mycelia and polysac-
charide by G. lucidum in shake flask cultures. The length of
carbon chain and the extent of unsaturation of fatty acids
determine the extent of stimulation or suppression. The
function was mainly explained in terms of membrane struc-
ture and it’s permeability. However, further studies are
required to understand the real mechanism.
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