Yield Performance of Ganoderma (1)

International Journal of Medicinal Mushrooms, 14(5): 521527 (2012)

5211521-1437/12/$35.00 2012 Begell House, Inc. www.begellhouse.com

Yield Performance of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (W.Curt.:Fr.) P. Karst. (Higher Basidiomycetes), Using Different Waste Materials as SubstratesMajid Azizi,1* Maryam Tavana,1 Mohammad Farsi,2 & Fatemeh Oroojalian3

1Department of Horticulture, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran; 2Department of Biotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran; 3Department of Life Science Engineering, Faculty of New Science and Technology, University of Tehran, Tehran, Iran

*Address all correspondence to: Majid Azizi, Department of Horticulture, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran; [email protected]

ABSTRACT: In this research the effect of sawdust, malt extract, and wheat bran on yield, biological efficiency (BE), and mycelia growth of Ganoderma lucidum was investigated. Three kinds of sawdust (beech, poplar, and hornbeam) as basal medium were mixed with two levels of wheat bran (5% and 10% w/w) and malt extract (2.5% and 5% w/w) as medium supplement for production of G. lucidum in factorial experiments on the basis of completely randomized design with three replications. The results showed that various kinds of sawdust affect fruiting body yield, BE, and mycelia growth rate significantly. The highest fruiting body yield and BE (102.58 g/kg and 12.89%, respectively) were found using hornbeam sawdust. The beech sawdust promotes the mycelia growth rate more than other sawdust. Analysis of variance showed that there is a significant interaction between the sawdust type and wheat bran, sawdust type and malt extract, and wheat bran and malt extract as far as yield and BE of G. lucidum was concerned. A final comparison of the different formulae indicated that the best combinations for high yield (142.44 g/kg) and BE (18.68%) were obtained in a combination of poplar sawdust with 5% malt extract and 10% wheat bran. The highest mycelia growth rate (10.6 mm/day) was obtained in a combination of beech sawdust with 2.5% malt extract and 10% wheat bran.

KEY WORDS: medicinal mushrooms, Ganoderma lucidum, sawdust, malt extract, wheat bran, biological efficiency

ABBREVIATIONS: ANOVA: analysis of variance; BE: biological efficiency; BS: beech sawdust; HS: hornbeam sawdust; ME: malt extract; PDA: potato dextrose agar; PS: poplar sawdust; RH: relative humidity; SSF: solid state fermentation; WB: wheat bran

I. INTRODUCTIONLingzhi or Reishi medicinal mushroom, Ganoder-ma lucidum (W.Curt.:Fr.) P. Karst. (Ganodermata-ceae, higher Basidiomycetes), has been used as a healthy food and medicine in the Far East for more than 2000 years.1 Ganoderma lucidum contains various active substances, including more than 140 different types of tri-terpenes and several types of polysaccharides.2 In China, Japan, Korea, and Taiwan, Lingzhi has been a popular folk or orien-tal medicine used to treat various human diseases, such as hepatitis, hypertension, hypercholesterol-emia, and gastric cancer. Recent studies on Ling-zhi have also demonstrated numerous biological activities, including antitumor, anti-inflammatory

effects, and cytotoxicity to hepatoma cells.2 In the traditional cultivation technique of G.

lucidum, it takes at least several months until fruit bodies are developed. This culture technique is used to obtain basidiocarp, which is used to make tonic or tea. The advantage of solid state fermentation (SSF) over other techniques is that a concentrated product can be obtained from a cheap substrate, such as an agricultural residue, with little pretreatment or en-richment.34 Supplements such as sucrose, wheat, and rice bran are generally added to the mix.5 Gon-zalez-Matute et al.6 reported that sunflower seed hull can be used as the main energy and nutritional source in the formulation of a substrate for cultiva-tion of G. lucidum in synthetic logs with an accept-

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522 International Journal of Medicinal Mushrooms

Yield Performance of Ganoderma lucidum usinG different Waste materials as substrates

content to approximately 60%70%. The mixture of lime (CaCO3) and gypsum (CaSO4H2O) (2%) in a ratio of 1:1 (w/w) and 2% KH2PO4 was thor-oughly mixed with the substrate. The substrates were poured into heat-resistant polypropylene bags (20 40 cm) with 1.0-kg capacity. The bags were plugged with cotton plugs using PVC rings and au-toclaved at 121C for 2 h. After cooling, the steril-ized bags were inoculated with 2% spawn (w/w) under a laminar air flow cabinet and incubated at 26 1C under dark conditions. The relative hu-midity (RH) was adjusted to 60%70% during the spawn run. When the mycelium had colonized the substrate completely, the bags were trans-ferred to a fruiting room at 22 1C, 80%90% relative humidity (RH) with a 5-h light exposure (200500 Lux) to promote fruit primordia. A stain-less steel blade was used to make a cross cut (+) on the bags. These +-shaped slits become the sites for fruiting body formation14 (Fig. 1). This process took 1420 d. In order to develop the primordia to stalk, we used a 12-h photoperiod (500700 Lux) at 26 1C. After 30 d, for cap formation light intensity was increased to 1500 Lux. Fruiting bod-ies were harvested according to Royse,15 when the caps become completely red and the white margin disappeared. Total yield (g/kg) was obtained from two flushes during the harvesting period. The BE percentage ([fresh weight of harvested mush-rooms/dry matter content of the substrate] 100) was calculated according to Royse.16

To determine the growth rate of mycelium, glass test tubes (18 mm 18 cm) were filled with substrates (medium for fruit production) and after sterilizing, were inoculated with one mycelial agar disc about 7 mm in diameter from the PDA cul-

able mushroom production rate, and the addition of 5% malt to sunflower seed hulls significantly improved mushroom productivity. Various sub-strates for G. lucidum cultivation have been inves-tigated.512 The suitability of rice bran, rice husks, coconut fiber, peanut hulls, corn, sorghum, and sugarcane bagasse as supplements for the substrate mixture was investigated by Triratana et al.13

The aim of the present study was to determine the best media for high yield, biological efficiency (BE), and mycelia growth rate using some sawdust and supplements such as wheat bran (WB) and malt extract (ME) for cultivation of G. lucidum on solid waste product. This investigation was per-formed on G. lucidum for the first time in Iran.

II. MATERIALS AND METHODSThe strain of G. lucidum was obtained from Re-search Group of Industrial Fungi Biotechnology, Ferdowsi University of Mashhad. The cultures were maintained on Potato Dextrose Agar (PDA) and stored at 4C. The mycelium was activated by growing on a PDA plate at 25C for 8 d. The my-celium from PDA was used for spawn production on wheat grains. For preparation of spawn, wheat grain was mixed with 1% CaSO4. Three kinds of sawdustbeech (Fagus orientals), poplar (Popu-las alba), and hornbeam (Carpinus betulus)was the basal medium, mixed with two levels of wheat bran (WB) (5% and 10% w/w) and ME (2.5% and 5% w/w) as medium supplement for fruit produc-tion of G. lucidum (Table 1). In our study we sieved the sawdust and used only the particles that passed through a 0.25-mm mesh, and the coarse WB after passing through US No. 16 screen was used.

The substrate was wetted to increased moisture

TABLE 1. Substrate Formulation Used for Fruit Bodies Production of Ganoderma lucidumWheat Bran(%)

Malt Extract(%)

Sawdust*(%)

5 2.55

88.586

10 2.55

83.581

*Three kinds of sawdust (beech, poplar, and hornbeam) were used as basal medium. Substrate combination was the same for three sawdust types. The rest (4%) were a mixture of lime (CaCO3) and gypsum (CaSO4H2O) (2%) in a ratio of 1:1 (w/w) and 2% KH2PO4.

523Volume 14, Number 5, 2012

Azizi et Al.

ture. The test tubes were incubated at 26 1C in the dark and mycelia radial growth was measured daily.

The experimental design was factorial experi-ments based on a completely randomized design with three replications. The data were analyzed us-ing the analysis of variance (ANOVA) and signifi-cant differences between means were determined by the multiple comparison test of Tukey with = 0.05.

III. RESULTS AND DISCUSSIONA. Effect of Sawdust TypeThe ANOVA showed that different kinds of saw-dust significantly (P < 0.05) affect yield, BE, and mycelia growth rate (Table 2). The mean compar-ison of the results showed that the highest yield and BE (102.58 g/kg and 12.89%, respectively) were found using hornbeam sawdust (HS), while the lowest yield and lowest BE (36.95 g/kg and 4%, respectively) were obtained when beech saw-dust (BS) was used (Fig. 2). The effect of sawdust type on mycelia growth rate also showed that the maximum and minimum mycelia growth rate was

obtained when BS and poplar sawdust (PS) were used as substrate, respectively. There was no sig-nificant difference between HS and PS (Table 2) as far as mycelia growth was concerned.

Yang et al.11 reported that a mixture of sawdust (80%) and stillage grain (20%) at a water content of 60% was optimal for the formation of fruiting bodies of G. lucidum. Also in another study, a saw-dust-based substrate supplemented with 20% tea waste was found to be the most suitable for high yield and BE.17 These findings are in conformity with studies that reported hardwood sawdust has been preferred for commercial production of G. lucidum.15,18

B. Effect of Wheat Bran Our results showed that the ratio of wheat bran significantly (P < 0.05) affected yield and BE of the mushroom, but wheat bran had no significant effect on growth rate (Table 3). The highest yield (78.85 g/kg) and BE (9.91%) were obtained when WB was used at 5% w/w, while the highest mycelia growth rate was achieved with 10% w/w (Table 3).

The nutritional value of substrates can be im-proved by nitrogen (N) supplementation.19 High levels of N supplementation should result in higher yields.11 Although N increases mushroom yield, above a certain level it inhibits fruiting of the mush-room.20 Higher doses of N-rich supplements re-sulted in temperature increases sufficient to kill the mycelia.19,21 Our results showed that wheat bran is a suitable material for supplementation of the sub-strate for production of G. lucidum fruiting bodies.

Supplements such as sucrose, wheat, and rice bran are generally added to the substrate mixture.5 Triratana et al.13 stated that rice bran, ground corn, and ground sorghum have provided the best my-celia growth and mushroom yield when added to the substrate mixture. Several researchers15,2223 also preferred rice bran to wheat bran for substrate supplementation, but in our country there is a lot

TABLE 2. The Simple Effect of Various Types of Sawdust on Yield, BE, and Mycelia Growth Rate of Ganoderma lucidumSawdusttype

Yield(g/kg)

BE(%)

Growth rate(mm/day)

HS 102.58 a* 12.89 a* 9.1 b*BS 36.95 c 4.68 c 10.0 aPS 80.39 b 10.29 b 9.0 bHS: hornbeam sawdust; BS: beech sawdust; PS: poplar sawdust.*Means in each column with different letters are significantly different (P < 0.05).

FIGURE 1. The cross cut (+) on colonized bags and formation of fruiting bodies.



of wheat bran with a low price in comparison to rice bran.

C. Effect of Malt Extracts Two levels of ME (2.5% and 5% w/w) significant-ly (P < 0.05) improved yield and BE but had no significant effect on growth rate (Table 4).

The highest yield (78.42 g/kg), BE (10.01%), and mycelia growth rate (9.6 mm/day) were ob-tained when the fruiting substrates were enriched with 5% ME. Increasing the level of ME from 2.5% to 5% increased all measured traits such as yield, BE, and mycelia growth rate of G. lucidum (Table 4).

There are a few reports on the effect of ME on fruiting of G. lucidum. Our findings are also in agreement with Gonzalez-Matute et al.,6 who re-ported that sunflower seed hull can be used as the main energy and nutrient source in the formulation of a substrate for the cultivation of G. lucidum, and the addition of 5% ME to the substrate improved mushroom mycelia growth rate.

D. Interaction of Sawdust Type and Wheat BranThe results showed that different substrate formu-lae significantly (P < 0.05) affect both yield and

BE, while mycelia growth rate was not affected significantly (Table 5).

The best treatment for getting the highest yield (132.09 g/kg) and BE (16.59 %) was horn-beam sawdust supplemented with 5% wheat bran (HS:5%WB), but the highest mycelia growth rate (10.4 mm/day) was produced in substrate contain-ing beech sawdust enriched with 10% wheat bran (BS:10%WB). The combination of PS:10%WB produced the lowest yield (35.36 g/kg) and BE (4.53%); the lowest mycelia growth rate (8.8 mm/day) was found in PS:5%WB.

According to our findings, increasing the level of WB increased mycelia growth rate but decreased the BE of G. lucidum. Lelly and Janssen19 also re-ported that high content of wheat bran decreased fruit production of the mushroom.

E. Interaction of Sawdust Type and Malt Extract (ME)There is a significant interaction (P < 0.05) be-tween sawdust type and ME as far as yield and BE of the mushroom is concerned, while sig-nificant interaction was not seen on the mycelia growth rate (Table 6). The maximum and mini-mum of yield (111.61 g/kg and 28.52 g/kg) and BE (14.02% and 3.67%) were obtained in HS:5%ME

TABLE 3. The Simple Effect of Two Levels of WB on Yield and BE of Gano-derma lucidumLevel of WB(w/w)

Yield(g/kg)

BE(%)

Growth rate(mm/day)

5 78.85 a* 9.91 a* 9.2 ns

10 67.76 b 8.67 b 9.6WB: wheat bran. *Means in each column with different letters are significantly different (P < 0.05).nsNot significant (P < 0.05).

FIGURE 2. The fruiting bodies of Ganoderma lucidum produced on hornbeam (A) and poplar (B) sawdust.

A B


Azizi et Al.

and BS:5%ME, respectively. We also found a significant difference between combinations of PS:2.5%ME with BS:2.5%ME and BS:5%ME.

Nasreen et al.24 investigated the effect of ME on mycelia growth of G. lucidum and found that medium containing ME improved mycelia growth of the mushroom.

F. Interaction of Various Levels of WB and METhe results showed that various levels of WB and ME significantly (P < 0.05) affect both yield and BE, while mycelia growth rate was not affected significantly (Table 7).

The results also showed that the maximum yield and BE were found with the combinations 5%WB:2.5%ME and 10WB:5%ME, although there is not a significant difference between 10%WB:5%ME and 5%WB:2.5%ME on yield and BE. The lowest yield and BE were detected in the combination 10%WB:2.5%ME. Therefore, the best and most economical combination for high yield and BE was 5WB:2.5%ME.

G. Interaction of Sawdust Type with Various Levels of WB and METhe analyzed data showed significant effects (P < 0.05) of the treatments on both yield and BE, while mycelia growth rates were not affected

significantly (Table 8). The comparison of the different formulae showed that the best combi-nations for high yield production (142.44 g/kg) and BE (18.68%) were PS:5%ME:10%WB fol-lowed by HS:5%ME:5%WB (135.86 g/kg and 17.07%) and HS:2.5%ME:5%WB (128.32 g/kg and 16.12%). The highest mycelia growth rate (10.6 mm/day) was obtained with the combination BS:2.5%M:10%WB.

The combination BS:5%ME:5%WB produced the lowest yield (22.3 g/kg) and BE (2.8%); also the lowest mycelia growth rate (8.1 mm/day) was found in the combination PS:2.5%ME:5%WB. Yield and BE of HS:5%ME:10%WB was not significantly different from that of HS:2.5%ME:10%WB and PS:52.5%ME:5%WB.

As a matter of fact, the mixtures containing BS produced lower yield and BE than the other combinations, although these mixtures showed the highest mycelia growth rate. These results suggest that the yield, BE, and mycelia growth rate were affected not only by the sawdust type, ME, or WB, but also by their combinations. Also, the mycelia growth rate is not a precise or sufficient criteria for comparison of medium mixtures in fruit produc-tion of the mushroom.

IV. CONCLUSIONSThe effects of various types of sawdust, levels of

TABLE 4. The Simple Effect of Two levels of ME on Yield, BE, and Mycelia Growth Rate of Ganoderma lucidumLevel of ME(w/w)

Yield(g/kg)

BE(%)

Growth rate(mm/day)

2.5 68.19 b* 8.57 b* 9.2 ns

5 78.42 a 10.01 a 9.6ME: malt extract.*Means in each column with different letters are significantly different (P



ME, and WB on the yield, BE, and mycelia growth rate of G. lucidum were investigated in this study. As described above, yield and biological efficiency of G. lucidum varied widely, depending on the type of sawdust, levels of WB, and ME, and also their combinations. Therefore, it is important to use the proper combination of substrate formulations for the commercial production of medicinal mush-

room G. lucidum fruiting bodies.

REFERENCES1. Fang QH, Zhong JJ. Submerged fermentation of higher

fungus Ganoderma lucidum for production of valuable bioactive metabolitesganoderic acid and polysaccha-ride. Biochem Eng J. 2002;10:6165.

2. Hsieh C, Yang F. Reusing soy residue for the solid-state fermentation of Ganoderma lucidum. Bioresour Technol.

TABLE 6. The Effect of Combinations of Sawdust Type and Two Levels of ME on Yield, BE, and Mycelia Growth Rate of Ganoderma lucidumSawdusttype

ME(w/w)

Yield(g/kg)

BE(%)

Growth rate(mm/day)

HS 2.55

93.55 a*111.61 a

11.75 a*14.02 a

9.1ns9.1

BS 2.55

45.37 c28.52 c

5.7 c3.67 c

10.19.9

PS 2.55

65.64 b95.14 a

8.25 b12.34 a

8.39.6

HS: hornbeam sawdust; BS: beech sawdust; PS: poplar sawdust, ME: malt extract. *Means in each column with different letters are significantly different (P < 0.05).nsNot significant (P < 0.05).

TABLE 7. The Effect of Combinations of Various Levels of WB and ME on Yield, BE, and Mycelia Growth rate of Ganoderma lucidum

WB (w/w) ME (w/w) Yield (g/kg) BE (%)Growth rate(mm/day)

5 2.55

89.03 a*68.67 b

11.18 a*8.63 b

8.8ns9.5

10 2.55

47.34 c88.18 a

5.94 c11.4 a

9.59.7

ME: malt extract, WB: wheat bran.*Means in each column with different letters are significantly different (P < 0.05).nsNot significant (P < 0.05).

TABLE 8. The Effect of Combinations of Sawdust Type, Various Levels of WB, and ME on Yield, BE, and Mycelia Growth Rate of Ganoderma lucidumWB(%)

ME(%) Sawdust type

Yield(g/kg)

BE(%)

Growth rate(mm/day)

5 2.55

HS 128.32 a*135.86 a

16.12 a*17.07 a

8.8x9.2

10 2.55

HS 58.77 bcd87.35 b

7.38 bcd10.97 b

9.49.1

5 2.55

PS 84.01 bc47.84 de

10.55 bc6.01 de

8.19.5

10 2.55

PS 47.28 de142.44 a

5.94 de18.68 a

8.59.7

5 2.55

BS 54.77 cde22.3 e

6.88 cde2.8 e

9.79.7

10 2.55

BS 35.97 de34.75 de

4.53 de4.52 de

10.610.2

HS: hornbeam sawdust; BS: beech sawdust; PS: poplar sawdust.*Means in each column with different letters are significantly different (P < 0.05). nsNot significant (P < 0.05).


Azizi et Al.

2004;91:1059.3. Wagner R, Mitchell DA, Sassaki GL, Amazonas MALA,

Berovic M. Current techniques for the cultivation of Ganoderma lucidum for the production of bimass, ganod-eric acid and polysaccharides. Food Technol Biotechnol. 2003;41(4):37182.

4. Erkel E. The effect of different substrate mediums on yield of Ganoderma lucidum (Fr.) Karst. J Food Agricult Environm. 2009;7(34):84144.

5. Chen HM. Reutilization of waste materials from a rice distillery for the cultivation of Ganoderma lucidum. MSc thesis, Tunghai University, Taiwan. 1998.

6. Gonzalez-Matute R, Figlas D, Devalis R, Delmastro S, Curvetto N. Sunflower seed hulls as a main nutrient source for cultivating Ganoderma lucidum. Micol Apli-cada Int. 2002;14:1924.

7. Wang MX, Gao XL. Study on substrates for Ganoderma lucidum Karst. and the key to high-yield cultivation man-agement. Edible Fungi China. 1990;1:1718.

8. Li Mi, Sun LM, Wang LZ. Study of the culture of Gano-derma lucidum with waste residue drawed furfural. J He-bei Agric Uni. 1997;20:4144.

9. Cha DY, Yoo YB. Cultivation techniques of Reishi (Ganoderma lucidum). Food Rev Int. 1997;13:37378.

10. Ji H, Wang Q, Wang H, Chen WJ, Zhu ZH, Hou H, Zhang W. Preliminary research on Flammulina velutipes and Ganoderma lucidum cultivation using maize straw. Edible Fungi China. 2001;20(6):1112.

11. Yang FC, Hsieh C, Chen HM. Use of stillage grain from a rice-spirit distillery in the solid state fermentation of Gano-derma lucidum. Process Biochem. 2003;39(1):2126.

12. Tiwari CK, Meshram PB, Patra AK. Artificial cul-tivation of Ganoderma lucidum. Indian Forester. 2004;130:105759.

13. Triratana S, Thaithatgoon S, Gawgla M. Cultiva-

tion of Ganoderma lucidum in sawdust bags. ISMS. 1991;13(2):Article 20.

14. Stamets P. Growing gourmet and medicinal mushrooms. 3rd ed. Berkeley, CA, USA: Ten Speed Press, 2000; p. 35569.

15. Royse DJ. Specialty mushrooms. Arlington, VA, USA: ASHS Press, 1996; p. 464475.

16. Royse DJ. Effect of spawn run time and substrate nutri-tion on yield and size of the shiitake mushroom. Mycolo-gia. 1985;77:75662.

17. Peksen A, Yakupoglu G. Tea waste as a supplement for the cultivation of Ganoderma lucidum. World J Micro-biol Biotechnol. 2009;25:61118.

18. Smith JE, Rowan NJ, Sullivan R. Medicinal mush-room: a rapid developing area of biotechnology for cancer therapy and other bioactivities. Biotechnol Lett. 2002;24:183945.

19. Lelley JI, Janssen A. Productivity improvement of oyster mushroom substrate with a controlled release of nutrient. Mushr News. 1993;41:613.

20. Zadrazil F, Brunnert H. Influence of ammonium nitrate on the growth and straw decomposition of higher fungi. Zeit Pflanzenernaehr Bodenkd. 1979;142:44655.

21. Rinsanka T. Cultivation technique of edible fungus, Gri-fola frondosa (Fr.) S.F. Gray (Maitake no Saibaiho). J Hokkaido Forest Products Res Inst. 1980. p. 1314.

22. Oei P. Mushroom cultivation. Leiden, The Netherlands: Backhuys Publishers, 2003.

23. Tham LX, Matsuhashi S, Tamikazu K. Growth and fruit body formation of Ganoderma lucidum on media supple-mented with vanadium, selenium and germanium. My-cosci. 1999;40:8792.

24. Nasreen Z, Kausar T, Nadeem M, Bajwa R. Study of dif-ferent growth parameters in Ganoderma lucidum. Mycol Aplicada Int. 2005;17(1):57.

Documents

Yield Performance of Ganoderma (1)