สาขาวิทยาศาสตร์และเทคโนโลยี Veridian E-Journal, Science and Technology Silpakorn University

ปีที่ 3 ฉบับที่ 6 เดือนพฤศจิกายน–ธันวาคม 2559 Volume 3 Number 6 November – December2016 ISSN 2408 - 1248

Cultivation of Lingzhi Mushroom, Ganoderma lucidum,

by Using Sugarcane Bagasse
*
Nopphawan Ninluam
Watcharapong Potiprasert*
Urarux Romreun*
**
Eakaphun Bangyeekhun

Abstract
Efficiency of sugarcane bagasse used as substrate on cultivation of Linzhi mushroom,
Ganoderma lucidum in comparison with sawdust was investigated. The effects of these
substrate on growth, cellulace activity and mushroom yield were analyzed. Growth of
G. lucidum strains in sawdust was faster than in sugarcane bagasse. G. lucidum strains grown in
sugarcane bagasse showed higher cellulase activity than in sawdust. Biological efficiency of
G. lucidum strain cultured in sugarcane bagasse substrate was higher than in sawdust,
indicating that the sugarcane bagasse could be substitute of sawdust substrate for G. lucidum
production.

Keyword: Lingzhi, Ganoderma lucidum, Mushroom cultivation, Sugarcane bagasse

Introduction
Lingzhi mushroom, Garnoderma lucidum (Fr.) Krast, has been use as a traditional
medicine to improve health and prolonging life in East Asia including China, Japan and Korea
for over 2,000 years (Zhou et al., 2012). Nowadays, several bioactive compounds regarding to
therapeutic properties from Lingzhi have been discovered. For instance, several types of
polysaccharide exhibited anticancer, antitumor, antiviral and antioxidant activity and able to
activate immune system. Triterpenoids, unsaturated hydrocarbon namely Ganoderic acids,
showed cytotoxicity against cancer cells and are considered to be anticancer agents. Besides,

*
Department of Microbiology, Faculty of Science, Silpakorn University, Nakhon Pathom 73000, Thailand
**
Department of Microbiology, Faculty of Science, Silpakorn University, Nakhon Pathom 73000, Thailand
Corresponding Author: [email protected]

390
Veridian E-Journal, Science and Technology Silpakorn University สาขาวิทยาศาสตร์และเทคโนโลยี
Volume 3 Number 6 November – December2016 ISSN 2408 - 1248 ปีที่ 3 ฉบับที่ 6 เดือนพฤศจิกายน–ธันวาคม 2559

they revealed antiviral, antioxidant and antimetastatic activity, as well as affected on lower
urinary tract symptoms (Bishop et al., 2015).
Since Lingzhi has a huge demand as a because of medicinal and cultural significance,
mushroom in nature is certainly not enough. Therefore, cultivation of Lingzhi is necessary.
Cultivation of mushroom is considered to be sustainable development by reason of organic
wastes are normally used as a substrate to produced food without damaging the environment.
Techniques for mushroom cultivation have been developed. Lingzhi can be cultivated using
both log and bag culture technique. In Thailand, Lingzhi is generally produced by bag culture
technique using a Para rubber wood sawdust as main substrate. Cultivation of mushroom
required the sawdust from the Southern of Thailand. As fuel is used for transportation, price
of the sawdust is increased according to distance from source to destination. The used of
local organic waste for mushroom cultivation is an alternative to save cost. There are numbers
of organic waste were reported for mushroom cultivation. These are included straw, husk,
water hyacinth, banana pseudo-stem, corn cob, cotton seed hull, coffee bean hull, spent beer
grain and sugarcane bagasse.
Sugarcane bagasse is a waste left from sugar industry as well as sugarcane juice mart.
The waste has been used as a fuel for electricity generation or raw material for fertilizer. It
would be better if they are used as a substrate for mushroom cultivation to produce such a
food before converted to electricity or fertilizer. Therefore, the aim of this study was to
evaluate growth, cellulase activity and yield of Lingzhi when sugarcane bagasse was used as a
substrate for mushroom cultivation.

Materials and Methods

1. Mushroom strains and cultivation
Ganoderma lucidum (Fr.) Krast strain A1, G2, DOA, G45, G5A, G5Z, HHK and SUN were
kindly provided by Dr. Surapol Rukpathum. The mushroom mycelia were maintained on
potato dextrose agar (PDA) by periodic subculturing.
2. Mycelial growth rate
To determine mushroom mycelium growth rate, a 5mm diameter agar plug of each
mushroom strain was inoculated to a PDA plate. Then, the plates were incubated at 30 C.
Diameter of mushroom colony was measured daily. Growth rate was calculated by size of
diameter of colony (mm.)/day.

391
สาขาวิทยาศาสตร์และเทคโนโลยี Veridian E-Journal, Science and Technology Silpakorn University
ปีที่ 3 ฉบับที่ 6 เดือนพฤศจิกายน–ธันวาคม 2559 Volume 3 Number 6 November – December2016 ISSN 2408 - 1248

3. Mushroom cultivation
To make mushroom grain spawn, sorghum grains were washed, submerged overnight
and boiled. Then, damp sorghum grains were filled in bottle and sterilized by autoclave.
The mushroom mycelium was transferred to sorghum grains and incubated at 30 C until the
grains were fully covered by mycelium.
A Para rubber wood sawdust and find chopped sugarcane bagasse were used as
main substrate for mushroom cultivation. The mushroom substrate was prepared mixing of 10
kilograms of either sawdust or sugarcane bagasse, 300 gram of rice bran, 10 gram of lime
(Calcium oxide), 20 gram of sodium sulfate, 100 gram of gypsum salt (magnesium sulfate) and
70% moisture content of water. 900 gram of substrate was packed in polypropylene bag and
sterilized by autoclave. After cooling down to room temperature, 10 gram of grain spawn was
transferred to each bag and incubated at room temperature until the bags were fully
colonized by mycelium. Colonization period of mycelium in substrate bags was recorded.
Then, the mushroom was induced by transfer the bags to chamber at 303 C and 80-90%
RH. Fruiting bodies were collected after 50 days. Mushroom productivity was expressed as
biological efficiency (fresh weight of mushroom/ dry weight substrate used  100).
4. Cellulase activity assay
To assay cellulase activity, 50 gram of substrate colonized by mushroom mycelium
was mixed with 100 ml of 0.2 M sodium acetate buffer pH 4.5 and shaken at 180 rpm for
1 hour. Then, the culture filtrate was obtained by filtration and centrifugation at 4 C, 8000
rpm for 10 minutes. 0.25 ml of culture filtrate was mixed with 0.25 ml of 1% Carboxymethyl
cellulose (CMC) in 0.05 M sodium acetate buffer, pH 4.5. For blank, 1% of CMC was added
after incubation. The reactions were incubated at 50 C for 50 minutes. The cellulase activity
was determined by using DNSA method. One unit (U) of enzyme activity was defined as the
amount of enzyme that produced 1 µmol of reducing sugar per minute under the conditions
assayed.
5. Statistical analysis
The experiment was set-up in triplicate. The data was analyzed by Analysis of
Variance (ANOVA) and Turkey’s Test in SPSS software.

392
Veridian E-Journal, Science and Technology Silpakorn University สาขาวิทยาศาสตร์และเทคโนโลยี
Volume 3 Number 6 November – December2016 ISSN 2408 - 1248 ปีที่ 3 ฉบับที่ 6 เดือนพฤศจิกายน–ธันวาคม 2559

Results and Discussion

The mycelium of G. lucidum strains were inoculated on potato dextrose agar (PDA)
and incubated at 30 C. The growth rate of mycelium was determined by measurement of
colonial diameter daily. The growth rate of G. lucidum strains could be divided into 6 groups.
G. lucidum strain G2 was the most grown, while strain G5Z was the least (Fig. 1). The different
growth characteristics found in these strains may be due to genetically difference
(Sawetsuwannakun and Bangyeekhun, 2011).

70 a
Mycelial growth rate

60 b c
50 c
(mm./day)

d
40
30 e e
20
f
10
0
A1 G2 DOA G45 G5A G5Z HHK SUN
G. lucidum strain

Figure 1. Growth rate of G. lucidum strains on PDA at 30 C for 5 days. The values represent
means of triplicate culture. Error bars indicate SD. The alphabet a-f shows the statistic
different.

Cultivation of G. lucidum strains was performed using bag culture. The sawdust and
sugarcane bagasse were used as main substrates for mushroom cultivation. Colonization period
of mycelium in substrate bags was determined. Growth of G. lucidum strains in sawdust
substrate was faster than in sugarcane bagasse substrate. G. lucidum strain A1 took 32-34 days
for fully colonization in sawdust or sugarcane bagasse bags. Whereas strain G2 ran through 52
days in bag containing sugarcane bagasse. Strain G2, G45, G5A and G5Z spent time in sugarcane
bagasse more than in sawdust substrate, while strain A1 was vice versa (Fig. 2).
Composition of cellolose, lignin and nitrogen in substrate could be influence on
colonization period in substrate. The sawdust contains cellulose and lignin with 58% and 41%
respectively (Sornprasert and Aroonsrimorakot, 2014) whereas, sugarcane bagasse comprises of
those with 44% and 24%, respectively (Pereira et al., 2011). Mycelial growth of G. lucidum was
efficient when cultured in high proportion of cellulose and lignin substrate (Sornprasert and
Aroonsrimorakot, 2014). Moreover, it was suggested that the spawn running in high nitrogen
393
สาขาวิทยาศาสตร์และเทคโนโลยี Veridian E-Journal, Science and Technology Silpakorn University
ปีที่ 3 ฉบับที่ 6 เดือนพฤศจิกายน–ธันวาคม 2559 Volume 3 Number 6 November – December2016 ISSN 2408 - 1248

substrate could be slower than in low amount of nitrogen (Yang et al., 2013). Nitrogen
composition in sawdust is 0.30% while in sugarcane bagasse is 1.23%.
d
60 a d c d b e a d d
c a
Colonization period on
substrate bag (day) 50 f e e d e c f b e e c c
g f f e f g
d f f d d
40 g g
30
20
10
0
A1 G2 DOA G45 G5A G5Z HHK SUN
G. lucidum strain

Figure 2. Colonization period of mycelium of G. lucidum strains in sawdust (whirt) or sugarcane

bagasse (black) substrate. The values represent means of triplicate culture. Error bars indicate
SD. The alphabet a-g shows the statistic different.

Total enzymes were extracted form mushroom cultivation bags containing either
sawdust or sugarcane bagasse as the substrate. Cellulase activities were assayed by using DNSA
method. The results revealed that cellulase activities of G. lucidum strain A1 G2 DOA G45 and
G5Z grown in sugarcane bagasse substrate were higher than others (Fig. 3).
Analysis of G. lucidum cultivated in sugarcane bagasse revealed that the mushroom
able to produced several lignocellulolytic enzymes, i.e. cellulases, hemicellulases and lignin
degrading enzymes (Manavalan et al., 2012). In other mushrooms, prophenol oxidase and
manganese peroxidase, the lignin degrading enzymes, were produced first and celluolytic
enzymes were produced afterward in sugarcane bagasse degradation (Dong et al., 2013). A
number of studies revealed that cellulase efficiency in cellulose hydrolysis is usually reduced
in the presence of lignin. This is probably due to the association cellulose with lignin, the
access blocking of enzyme to cellulose, and the unproductive binding of the enzymes to lignin
(Vermaas et al., 2015). Composition of lignin in sawdust and sugarcane bagasse is 41% and
24% respectively. Therefore, it is possible that low cellulase activity of G. lucidum grown in
sawdust was a result of high percentage of lignin in the substrate.

394
Veridian E-Journal, Science and Technology Silpakorn University สาขาวิทยาศาสตร์และเทคโนโลยี
Volume 3 Number 6 November – December2016 ISSN 2408 - 1248 ปีที่ 3 ฉบับที่ 6 เดือนพฤศจิกายน–ธันวาคม 2559

a a
0.80 a a b b
b b

Cellulase activity (Unit)

0.70 b b c b c
b c
0.60 c c aa d c c d
c
0.50 d d e d d c e
d
0.40 e e f e e d f
f f f e d
0.30 e
0.20
f e
f
0.10 f f f f
0.00
A1 G2 DOA G45 G5A G5Z HHK SUN
G. lucidum strain

Figure 3. The cellulase activity of G. lucidum strains grown in sawdust (white) or sugarcane
bagasse (black) substrate. The values represent means of triplicate culture. Error bars indicate
SD. The alphabet a-f shows the statistic different.

Efficiency of substrate on yield of G. lucidum fruiting body was determined.Biological

efficiency of G. lucidum strain G2, DOA, G45, G5A, G5Z and HHK cultured in sugarcane bagasse
substrate was statistically higher than in sawdust substrate (Fig. 4).
It was suggested that C:N ratio of the substrate could influence on growth and
development of mushroom. Condition with high C:N ratio was suitable for the mycelium
growth, whereas low C:N ratio was appropriate for primodia and mushroom development
(Yang et al., 2013). Substrate with C:N ratio of 70-80 yielded proper mushroom fruiting body,
while with C:N ratio of 50 generated only the strip without cap and C:N ratio of lower than
50 gave no fruiting body formation. In this study, the C:N ratio was not determined in the
substrates. Therefore, it could be merely speculated that C:N ratio in sugarcane bagasse was
higher than that in sawdust and influenced on fruiting body production of G. lucidum.
Sugarcane bagasse showed potential substrate for the mushroom production of many species,
such as Pleurotus ostreatus, Lentinus edodes (Vetayasuporn et al., 2006; Salmones et al.,
1999). According to this study, sugarcane bagasse could be substitute of sawdust substrate for
G. lucidum production.
Yield of mushroom could be influenced from several factors. For instance, amount
of appropriate nutrient in substrate would provide energy for mycelial growth and mushroom
production. Physical structure of substrate would increase water-absorbing capacity (Yang et
al., 2013). Hence, combination of several agricultural waste could make the best substrate
formula for mushroom production. In future study, we will shed the light on finding the proper
mixture of ingredients form several agricultural wastes in order to increase in mushroom yield.
395
สาขาวิทยาศาสตร์และเทคโนโลยี Veridian E-Journal, Science and Technology Silpakorn University
ปีที่ 3 ฉบับที่ 6 เดือนพฤศจิกายน–ธันวาคม 2559 Volume 3 Number 6 November – December2016 ISSN 2408 - 1248

Biological efficiency (%)

12 c a
a a
10 d c a c a
c e b
d b d b
8 d f c e e
c c
e d d f
6 d
f e e e
4 f f f
2 f
f
0
A1 G2 DOA G45 G5A G5Z HHK SUN
G. lucidum strain

Figure 4. Biological efficiency of G. lucidum strains grown in sawdust (white) or sugarcane

bagasse (black) substrate. The values represent means of triplicate culture. Error bars indicate
SD. The alphabet a-g shows the statistic different.

Conclusion
Mushroom cultivation is one of the effective means to convert agricultural waste to
food. Beside sawdust, several agricultural wastes such as rice straw, banana leaves, corn cob
and sugarcane bagasse can be used as an alternative substrate for mushroom cultivation.The
present study reveals that sugarcane bagasse is useful material for cultivation of Lingzhi
mushroom, a medicinal mushroom. Therefore, cultivation of Linzhi by using a cheap substrate
like sugarcane bagasse may be a profitable agribusiness for making an extra income.

Acknowledgements
We would like to thank the Department of Microbiology, Faculty of Science,
Silpakorn University for financial support.

References
Bishop, K.S., Kao, C.H.J., Xu, Y., Glucina, M.P. and Paterson, R.R.M. (2015). “From 2000 years of
Ganoderma lucidum to recent developments in nutraceuticals.”
Phytochemistry. 2015; 114 : 56-65.
Dong, X.Q., Yang, J.S., Zhu, N., Wang, E.T. and Yuan H.L. (2013). “Sugarcane bagasse degradation
and characterization of three white-rot fungi”Bioresource Technology.131 : 443-451.
Manavalan, T., Manavalan, A., Thangavelu, K.P. and Heese, K. (2012). “Secretome analysis of
Ganoderma lucidum cultivated in sugarcane bagasse.” Journal of Proteomics. 77 :
298-309.

396
Veridian E-Journal, Science and Technology Silpakorn University สาขาวิทยาศาสตร์และเทคโนโลยี
Volume 3 Number 6 November – December2016 ISSN 2408 - 1248 ปีที่ 3 ฉบับที่ 6 เดือนพฤศจิกายน–ธันวาคม 2559

Pereira, P.H.F., Voorwald, H.C.J., Cioffi, M.O.H., Mulinari, D.R., Da Luz, S.M. and Da Silva, M.L.C.P.
(2011). “Sugarcane bagasse pulping and bleaching: Thermal and chemical
characterization”. BioResource. 6 : 2471-2482.
Salmones, D., Mata, G., Ramos, L.M. and Waliszewski, K.N. (1999). Cultivation of shiitake
mushroom, Lentinus edodes, in several lignocellulosic materials originating from the
subtropics.” Agronomie. 19 : 13-19.
Sawetsuwannakun, K. and Bangyeekhun, E. (2011). “Physiological and genetics characterization
of Ganoderma lucidum strains” The 37th congress on Science and Technology of
Thailand at Centara Grand & Bangkok Convension Centre, Bangkok.10-12 October
2011. pp.1-5.
Sornprasert, R. and Aroonsrimorakot, S. (2014). “Utilization of Vetiveria zizaniodes (L.) Nash
leaves in Ganoderma lucidum cultivation” APCBEE Procedia. 8 : 47-52.
Vermaas, J.V., Petridis, L., Qi, X., Schulz, R., Lindner, B. and Smith, J.C. (2015). Mechanism of
lignin inhibition of enzymatic biomass deconstruction” Biotechnology for Biofuels.
8 : 217-232.
Vetayasuporn, S., Chutichudet, P. and Cho-Ruk, K. (2006). Bagasse as a possible substrate for
Pleurotus ostreatus (Fr.) Kummer cultivation for the local mushroom farms in the
Northern of Thailand.” Pakistan Journal of Biologial Science. 9 : 2512-2515.
Yang, W.J., Guo, F.L. and Wan, Z.J. (2013). “Yield and size of oyster mushroom grown on
rice/wheat straw basal subatrate supplemented with cotton seed hull.”
Saudi Journal of Biological Sciences. 20 : 333-338.
Zhou, X.W., Su, K.Q. and Zhang, Y.M. (2012). “Applied modern biotechnology for cultivation of
Ganoderma lucidum and development of their products.” Applied Microbiology
and Biotechnology 93 : 941–963.

397