Our Nature

O.K. Gurung, U. Budathoki and G. (2012)

Parajuli10: 191-198
/ Our Nature (2012) 10: 191-198

Effect of Different Substrates on the Production of

Ganoderma lucidum (Curt.:Fr.) Karst.

O.K. Gurung1*, U. Budathoki2 and G. Parajuli3

1
Shree Punya Higher Secondary School, Rajarani 6, Dhankuta
2
Central Department of Botany, T.U., Kirtipur, Kathmandu
3
Plant Pathology Division, NARC, Khumaltar, Lalitpur
*
E- mail: [email protected]

Abstract
In this study, the effects of various kinds of sawdust and supplements on the yield of
Ganoderma lucidum were investigated in artificial cultivation. The sawdust of Alnus
nepalensis, Shorea robusta and Dalbergia sisoo and supplements of rice bran , wheat
bran, corn flour and gram flour were used as substrates in Ganoderma lucidum
cultivation . Sawdust alone was used in control as a substrate media in the cultivation.
Significant differences (P< 0.05) were found among varieties of sawdusts and
supplements. Similarly significant differences (P<0.05) were found in sawdust with
supplements and without supplements in yield and biological efficiency. Dalbergia sisoo
sawdust could not give yield in ambient condition; Shorea robusta gave very low yield
and biological efficiency. Alnus nepalensis gave good yield and biological efficiency
compared to the other sawdust.The highest yield and Biological efficiency were obtained
from gram flour compared to the other brans. Alnus nepalensis sawdust supplemented
with gram flour showed higher yield among all treatments. Supplementation showed
positive role in mycelia growth and yield of mushroom.

Key words: Biological efficiency,suppliments, cultivation, Ganoderma lucidum, sawdust,

substrate, yield

Introduction
Mushrooms are the fruiting bodies of Ganoderma lucidum contains Triterpenes –
macrofungi. Mushroom cultivation has triterpenoids, polysaccharides, aminoacids ,
become one of the most profitable fungal immunomodulatory proteins and
agribusiness that could produce food steroids. Several triterpenes, eg. ganoderans
products from different substrates and help A,B and C, isolated from G.lucidum fruit
to dispose them in an environmentally bodies have been shown to have a strong
friendly manner (Bano et al., 1993). Red hypoglycemic effect (Chang and Miles,
mushroom (Ganoderma lucidum) is a 2004). All parts of Ganoderma lucidum ,
medicinal mushroom. Ganoderma lucidum such as spore, mycelium and basidiocarp,
(Curt.Fr.) P. Karst. ( Lingzhi in Chinese; are used for health purpose. For
Reishi, mannentake, or Sachitake in pharmaceutical purposes, a medicinal
Japanese; and Youngzi in Korian) is a mushroom were almost always prepared
species of Class Basidiomycetes, which either as hot water extracts, concentrate or
belongs to the family Polyporaceae or in powdered form (Smith et al., 2002).The
Ganodermataceae of the order amount of wild Ganoderma is inadequate
Aphylophorales ( Chang and Miles, 2004). and unsafe to consume as insects and snakes
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make the mushroom poisonous; its still practiced mainly in Asia, is on logs of
cultivation is essential to meet the demand. broad leaved hardwood trees. The rapid
In this connection, the techniques of worldwide cultivation of medicinal
cultivation have been developed and mushrooms is largely due to the use of
described during the last 15-20 years. polypropylene bags or containers.
Especially many workers have attempted to Ganoderma lucidum grows on a wide
obtain agent ingredients by extraction from variety of dead or dying trees, eg.
mycelium using submerged culture. Deciduous trees, especially Quercus, Acer,
Nowadays most of the medicinal Alnus, Betula, Pyrus, Magnolia, etc.
mushrooms have been subjected to large Currently, the methods most widely adopted
scale artificial cultivation by solid substrate for commercial production are the wood
or low moisture fermentation. Mushroom log, short wood segment, tree stump,
cultivation is the only large scale sawdust bag and bottle procedures (Wasser,
biotechnological process that creatively 2005).
utilizes lignocellulosics (Staments, 2000). For the cultivation of most medicinal
Ganoderma lucidum has been cultivated by mushrooms the basic substrate is hardwood
using several different substrates and by sawdust( a mixture of fine and coarse
maintaining growth parameters such as sawdust to ensure good aeration) 75-80%,
temperature, relative humidity, water supplemented with wheat bran 20% gypsum
content, air pH and light intensity (Chang 1%, sucrose 1%, moisture content 60-65%
and Miles, 2004). Ganoderma lucidum is and pH 5.5- 6.5. A successful artificial
traditionally cultivated in solid culture to cultivation has been reported on solid
obtain fruiting body or basidiocarp and substrates utilizing sawdust and different
spawn is prepared using substrates such as agricultural wastes (rice bran, wheat bran,
grain, saw dust or wood. The advantage of sugarcane bagasse, rice husks, coconut
SSF (solid state fermentation) over other fiber, peanut hulls, banana leaves etc). Tea
techniques is that a concentrated product waste was investigated by Peksen and
can be obtained from a cheap substrate, such Yakupoglu (2009), and it was concluded
as an agricultural residue with little that tea waste can be used as a supplement
pretreatment or enrichment (Wagner et for substrate preparation in G. lucidum
al.,2003). This method of production has cultivation. Erkel (2009) investigated the
also several disadvantages, including the effect of three kinds of saw dusts (poplar,
long time required to cultivate the fruiting oak and beech) and brans (Wheat, rice and
body of Ganoderma lucidum, the difficulty corn) on the yield of G. lucidum. The
of controlling the quality of the product, and highest yield and biological efficiency were
the ease with which the culture is affected obtained from oak sawdust compared to the
by the environmental changes. Recently, other sawdusts and also from wheat bran
submerged fermentation technology for compared to the other supplements.
mycelial cultures of G. lucidum has been In Nepal, G. lucidum had been
developed which none of the weaknesses cultivated in 2004 AD in Nepal Agricultural
has stated above (Yang and Lieu, 1998; Lee Research Council taking four different types
et al., 1999). The historical method of of substrate composition (sample A (saw
cultivation of medicinal mushrooms, and dust 90%, rice bran 10%), sample B
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(sawdust 72% , Corn meal 20%, rice bran four kinds of supplements including rice
7.8 %, CaCO3 0.2%), sample C (sawdust bran (RB), wheat bran (WB), corn flour
90%, Wheat bran 12%) and sample D( (CF), gram flour (GF). The sawdust was
sawdust 90% , wheat bran 10%). 65-70 % used with different supplements as well as
moisture was adjusted in all four samples. in controlled condition (without
The result showed that the above mentioned supplements) as artificial medium materials.
composition can be substrate for cultivation The sawdust and supplement were mixed at
of G. lucidum (Plant Pathology Division, a ratio of 9:1 based on their dry weight
2007). Similarly, paddy straw was used in (W/W). No chemical constituents of media
Plant Pathology Division Nepal Agricultural used in the fifteen treatments were
Research Council) and the result showed determined. Moisture content of mixtures
that paddy straw is also one of the substrate was adjusted approximately 60-65 % with
for cultivation of Ganoderma lucidum. In the help of moisture analyzer and palm test
the same year two strains (Japanese strain method before sterilization (Kwon and Kim,
and Philippines strain) of G. lucidum were 2004). No CaCO3 and gypsum were added
introduced for studying on log of Alder to stabilize pH of the substrates. The pH of
(Alnus nepalensis) and found biological each treatments were measured with litmus
efficiency of Japanese strain was higher paper and all showed approximately 5.5-
than that of Philippines strain. However 6.5.
after the first flush the logs inoculated with The substrates were packed into the
Japanese strain were contaminated with polypropylene bags and plugged with cotton
other pathogens. Hence, the experiment plug. Then the substrate bags were
needs to conduct in next time to conform the autoclaved at 1210C for 1.5 hrs. After
efficiency of these two strains in Alder logs cooling, sterilized bags were inoculated in
(Plant Pathology Division, 2010). the laminar air flow cabinet and incubated
in dark incubation room. The temperature of
Materials and methods the incubation room was maintained with
The research activities of the present study the help of fan. The temperature was
were carried out in the laboratory of Plant recorded during the incubation period
Pathology Department of Nepal Agricultural regularly and was 28- 300C. The substrates
Research Council, Khumaltar lalitpur Nepal. were kept in the incubation room till full
The sawdusts were collected from different colonization of the mycelium. When the
saw mill of Lalitpur district and mycelium had colonized on the substrate
supplements were collected from rice mills completely, the bags were shifted to
and other shops. The spawn of Ganoderma cropping room [ room having high temper-
lucidum was brought from Centre for ature i.e 30-320C, high humidity and light
Agricultural Technology (CAT), Imadole,
exposure] for the formation of fruiting
Lalitpur, Nepal. The study was conducted in
June to September 2010. bodies. The mouth of the bags were opened
The experiment was carried out with and kept on metal tray containing water to
three kinds of sawdust including Alnus make high humidity. In growth stage
nepalensis (AS), Shorea robusta (SS), frequent watering should be done in order to
Dalbergia sisoo (DS), in control and with raise relative humidity approx. 80-90%.

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Primordial formation of Ganoderma matured in 20 days in both Shorea robusta

lucidum initiated in different days in and Alnus nepalensis sawdust except for
different treatments. Fruiting bodies were Alnus nepalensis sawdust in control 17
harvested when the caps became completely days.
red and the white margin disappeared. The The average first harvest days for
total yield for each treatment was measured Shorea robusta were found 66 days. The
from two flushes in a harvest period of 75 average first harvest of Alnus nepalensis
days. The colonization period, primordial sawdust with different supplement and
formation days and first harvest period were control were found 60 days. Alnus
recorded and compared for all treatments. nepalensis showed shorter period than that
The biological efficiency (BE) percentage of Shorea robusta saw dust (Fig.3). In this
of fresh weight of harvested mushrooms/dry experiment, the colonization period,
matter content of the substrate × 100 was primordial formation period and first
calculated (Royse, 1985). Experimental harvest days for Dalbergia sisoo could not
design was a Completely Randomized be obtained due to very poor mycelial
Design (CRD) with four replicates. The data growth.
were analyzed by using the analysis of
variance (ANOVA) and group means were Effect of different type of sawdust on yield
compared by Duncan Multiple Range Test and BE
(DMR) using the SPSS computer program Sawdust is the main ingredient used in
(Version 16.0). substrate mixtures for Ganoderma lucidum
cultivation. To investigate the feasibility of
Results and discussion using three kinds of sawdusts as basal
substrates, Shorea robusta, Dalbergia sisoo
Colonization period, primordial formation and Alnus nepalensis sawdust were tested
and first harvest days with wheat bran, gram flour, rice bran, corn
There was equal colonization period (35 flour as supplements and sawdust alone.
days) of Shorea robusta saw dust The highest yield of 15.05 g/400 gm and BE
supplemented with gram flour, wheat bran, of 15.69 % were obtained from Alnus
corn flour and rice bran (Fig.1). The nepalensis sawdust, and followed by Shorea
colonization period of Shorea robusta in robusta (1.5 g/400gm and 0.512%).
control could not be obtained due to slow Dalbergia sisoo could not give any yield
mycelial growth and it was contaminated due to very poor mycelial growth (sawdust
with other mould and discarded (Fig. 1). of Dalbergia sisoo was very fine as a result
The average primordial formation there was aeration problems). There were
period for Shorea robusta sawdust with significant differences (P<0.05) among
supplements was longer (46.0 days) than Alnus nepalensis saw dust and Shorea
that of Alnus nepalensis. The primordial robusta sawdust. The Shorea robusta could
formation period for Alnus nepalensis in not give good yield and BE was very low.
control was found 43 days. The Biological Efficiency of Alnus
The primordial formation period for nepalensis is agreement with Erkel (2009).
Alnus nepalensis with supplements were He found BE (17.48%) from oak sawdust,
found 40 days (Fig. 2). The primordia (15.94%) from Beach sawdust and (15.09%)
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from poplar sawdust. BE of Shorea robusta supplements, gram flour was not different
was inconsistent with Erkel (2009). This from Rice bran statistically. Similarly wheat
may be due to tight packing and poor bran, corn flour and rice bran were not
aeration and low water holding capacity of different statistically. This result was
fine sawdust because the saw dust of contrast to result obtained by (Erkel, 2009).
Dalbergia sisoo and Shorea robusta was He found the highest yield (63.66 g/kg)
finer than that of Alnus nepalensis. The from wheat bran followed by corn bran
difference may also be due to difference in (52.37 g/kg). Rice bran gave the lowest
nutritional quality of the different saw dust. yield among bran (45.17 g/kg). He used
The findings are in conformity with some 20% of bran but in this study 10% bran was
authors who reported broad leaved hard used. This contrast seems to be resulted
wood sawdust was preferred for commercial from the ratio of supplements. Gram flour
production. But sawdust must be coarse and showed significant with rice bran, wheat
coarse sawdust or wood chips must be bran and corn flour in biological efficiency.
mixed in fine sawdust to increase aeration Although biological efficiency showed
and water holding capacity. As a result difference among the supplements, wheat
mycelium can spread easily and densely. bran was not different from corn flour
Different letters along the column statistically.
indicate significant differences of the mean These results suggest that the yield and
(p=0.05) according to Duncan Multiple biological efficiency are different for
Range Test. different substrates.
Different letters along the column
Effect of different type of supplements and indicate significant differences of the mean
control on the yield and biological (p=0.05) according to Duncan Multiple
efficiency of Ganoderma lucidum Range Test.
Significant differences (P<0.05) were found
among various types of supplements and Conclusions
control (Table 1 and 2). The highest yield The effect of various kinds of sawdust and
(11.25 g/400 gm) and BE (12.19%) were supplements on the yield of Ganoderma
obtained from Gram flour followed by Rice lucidum was investigated in this study. As
bran (9.62g/400 gm) and 10.09%. Rice bran described above, yield and biological
was followed by wheat bran (8.5 g/400 gm efficiency of Ganoderma lucidum varied
and 7.27%. Corn flour gave lowest yield widely depending on the kind of sawdust
(7.0 g/400gm) and BE (6.9%) among and supplements. Therefore it is important
supplements. to use the proper substrate for the
Control (sawdust without supplements) commercial production of G. lucidum. Alnus
gave lowest yield (3.12 g/400 gm) and BE nepalensis sawdust supplemented with gram
(3.91). There were significant difference flour showed highest yield among all
between substrate with supplements and treatments. There was no significant
control in yield and BE. Gram flour showed difference between gram flour and rice bran
significance difference with Wheat bran and supplement. As rice bran is industrial
Corn flour in yield statistically. Although byproduct and economically cheaper than
yield showed different among the gram flour, it is recommended to use rice
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Figure1. Comparison of colonization period

Figure 2. Comparison of primordial formation period

Figure 3. Comparison of first harvest period

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Table 1. effect of various kinds of sawdust on yield and Biological Efficiency of Ganoderma lucidum
sawdust supplement Yield g/400gm mean Biological Mean
efficiency (%)
Shorea robusta Control 0.00a 0.00 a
a
sawdust Gram flour 1.75 1.75 a
(SS) Wheat bran 0.00 a 0.65 b 0.00 a 0.512 b
a a
Corn flour 0.00 0.00
Rice bran 1.5 a 0.81 a

Alnus nepalensis Control 6.25c 7.81 c

sawdust Gram flour 20.75 a 22.62 a
(AS) Wheat bran 17.0ab 15.05 a 14.58 b 15.69 a
Corn flour 14.0b 13.81 b
Rice bran 17.75 ab 19.37 a

Table 2. Effect of different type of supplement and control on yield and Biological efficiency of Ganoderma
lucidum
supplements Sawdust Yield (g/400gm) mean Biological mean
efficiency (%)
control SS 0.00 3.12 c 0.00 3.91d
AS 6.25 7.81
GF SS 1.75 11.25 a 1.75 12.19 a
AS 20.75 22.62
WB SS 0.00 8.50 b 0.00 7.27 c
AS 17.00 14.58
CF SS 0.00 7.00 b 0.00 6.90 c
AS 14.0 13.81
RB SS 1.5 9.62 ab 0.81 10.09 b
AS 17.15 19.37

bran as a supplement with A. nepalensis Chang, S.T. and, P.G. Miles 2004. Mushrooms:Cul-
sawdust to cultivate Ganoderma lucidum. tivation, Nutritional Value, Medicinal effect and
Environmental Impact (2nd edition) Boca Raton,
Wheat bran can be used as an alternative CRC press.
supplement. Supplementation showed Erkel, E.I. 2009. The effect of different substrate
positive role in mycelia growth and yield of medium on the yield of Ganoderma lucidum (Fr.)
mushroom. So, supplementation is required Karst. Journal of Food, Agriculture and
Environment 7(3-4): 841-844.
for commercial production of Ganoderma Kwon, H. and B.S. Kim 2004. Mushroom growers’
lucidum. Handbook1; Bag cultivation. In Oyster mushroom
Cultivation. Mushworld, Republic of Korea.
References pp.144-159.
Bano, Z., M.N. Shasirekhaandm and S. Rajarathnam Lee, K.M.., S.Y. Lee and H.Y. Lee 1999. Bistage
1993. Improvement of the bioconversion of control of pH for improving exopolysaccharides
biotransformation efficiencies of the oyster production from mycelia of Ganoderma lucidum
mushroom (Pleurotus sajor-caju) by supplement- in an air- lift fermenter. J.Biosci.Bioeng. 88(6):
ation of its rice straw with oil seed cakes. Enzyme 646-650.
and Microbial Technology 15: 985-989. Peksen, A. and G.Yakupoglu 2009. Tea waste as a
supplement for the cultivation of Ganoderma
197
 O.K. Gurung, U. Budathoki and G. Parajuli / Our Nature (2012) 10: 191-198

lucidum. World Journal of Microbiology and biotechnology for cancer therapy and other
Biotechnology 25(4): 611-618. bioactivities. Biotechnology Letters 24: 1839-
Plant Pathology Division (PPD) 2007. Cultivation 1845.
methodology of Red mushroom. In Annul Report Staments, P. 2000. Growing gourmet and medicinal
(2005-2006). Nepal Agricultural Research mushrooms (3rd edition). Ten Speed Press
Council. pp. 57-59. Olimpia, WA.
Plant Pathology Division (PPD) 2010. Study on Wagner, R., D.A. Mitchell, G.L. Sassaki, M.A.L.A.
cultivation of Red mushroom (Ganoderma Amazonas and M. Berovic 2003. Current
lucidum) on bed log of Uttis/Alder (Alnus techniques for the cultivation of Ganoderma
nepalensis). pp. 69. lucidum for the production of biomass, ganoderic
Plant Pathology Division (PPD) 2010. Study on acid and polysaccharides. Food Technol.
cultivation technology of Ganoderma spp. on Biotechnol. 41(4): 371-382.
paddy straw. In Annual Report 2008-2009. Nepal Wasser, S.P. 2005. Reishi or Ling Zhi (Ganoderma
Agricultural Research Council. pp. 66. lucidum). Encyclopedia of Dietary Supplements.
Royse, D.J. 1985. Effect of spawn run time and pp. 603-622.
substrate nutrition on yield and size of the Shiitake Yang, F.C. and C.B. Liau 1998. Effect of cultivating
mushroom. Mycologia 77: 756-762. conditions on the mycelial growth of Ganoderma
Smith, J.E., N.J. Rowan and, R. Sullivan 2002. lucidum in submerged flask cultures. Bioprocess
Medicinal mushrooms: A rapid developing area of Engineering 19: 233-236.

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