effects of cultivating conditions on the mycelial growth

Liau Abstract In this paper the effects of environmental con-ditions on the mycelial growth of Ganoderma lucidum were investigated in shake ¯ask cultures.. However, too high rotating spe

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; K2HPO40.5, KH2PO40.5, MgSO4 á 7H2O 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

5 mm ´ 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 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 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 nmyceli-umber 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

References

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Li, J.; Reishi: Ganoderma lucidum and Ganoderma tsugae: Bioactive substances and medicinal effects Food Reviews In-ternational, 11 (1) (1995) 151±166

2 Sone, Y.; Okuda, R.; Wada, N.; Kishida, E.; Misaki A: Structure and antitumor activities of the polysaccharide isolated from fruiting body and the growing culture of mycelium of Ga-noderma lucidum Agric Biol Chem 49 (9) (1985) 2641±2653

3 Tseng, T.C.; Shiao, M.S.; Shieh, Y.S.; Hao, Y.Y.: Study on Ganoderma lucidum 1 Liquid culture and chemical compo-sition of mycelium Bot Bull Academia Sinica 25 (1984) 149± 157

4 Litch®eld, J.H.: Submerged culture of mushroom mycelium In: Peppler, H J (Ed.) Microbial Technology, pp 107±144 New York: Reinhold Publishing Corporation 1967

5 Eyal, J.: Mushroom mycelium growth in submerged culture-potential food applications In: Goldberg, I.; Williams, R (Eds.) Biotechnology and Food Ingredients, pp 31±64, New York: Van Nostrand Reinhold 1991

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7 Liau, C.B.: M Sci thesis, Department of Chemical Engineering, Tunghai University, Taiwan, 1996

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)