Songklanakarin Journal of Science and Technology SJST R2 Seephueak. Journal: Songklanakarin Journal of Science and Technology

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1 Songklanakarin Journal of Science and Technology SJST--0.R Seephueak Effects of Palm Oil Sludge as a Supplement for Ganoderma lucidum (Fr.) Karst Cultivation Journal: Songklanakarin Journal of Science and Technology Manuscript ID SJST--0.R Manuscript Type: Original Article Date Submitted by the Author: 0-Sep- Complete List of Authors: Seephueak, Pornsil; Rajamangala University of Technology Srivijaya, Faculty of Agriculture Preecha, Chaisit; Rajamangala University of Technology Srivijaya, Plant Science Seephueak, Wuttichai; Rajamangala University of Technology Srivijaya, Animal Science Keyword: biological efficiency, lingzhi, mushroom cultivation, formula, waste

2 Page of Songklanakarin Journal of Science and Technology SJST--0.R Seephueak 0 Original Article Effects of Palm Oil Sludge as a Supplement for Ganoderma lucidum (Fr.) Karst Cultivation Pornsil Seephueak *, Chaisit Preecha, and Wuttichai Seephueak Division of Plant Science, Faculty of Agriculture, Rajamangala University of Technology Sivijaya, Thung Yai, Nakhon Si Thammarat, 00, Thailand Division of Plant Science, Faculty of Agriculture, Rajamangala University of Technology Sivijaya, Thung Song, Nakhon Si Thammarat, 00, Thailand Division of Animal Science, Faculty of Agriculture, Rajamangala University of Technology Sivijaya, Thung Yai, Nakhon Si Thammarat, 00, Thailand * Corresponding author, address: spornsil@gmail.com Abstract The objective of this study was to determine the optimum rate of palm oil sludge, for Ganoderma mushroom (Ganoderma lucidum (Fr.) Karst) cultivation. Different concentrations of palm oil sludge (-% by dry weight) were mixed with rubber sawdust and used to grow the Ganoderma mushroom in plastic bags. Growth rate of the mycelia, number of basidiocarp, cap diameter, thickness of pileus and yield were compared with the control (non-supplemented) and the % rice bran (standard formula). The results showed that used of % palm oil sludge was the optimum concentration for Ganoderma cultivation. Yield on g/bag of 00 kg rubber sawdust included % palm oil sludge, % Ca(OH) and 0.% Mg(SO ) was. g/bag (B.E.=.0%). Not significant when compared with % rice bran yield obtained. g/bag (B.E.=.%). Keywords: biological efficiency, lingzhi, mushroom cultivation, formula, waste

3 Songklanakarin Journal of Science and Technology SJST--0.R Seephueak Page of 0. Introduction Ganoderma lucidum (Fr.) Karst. is a basidiomycetes mushroom belonging to the family Polyporaceae. In Thailand and around the world, it is called by its Chinese names which are Lingzhi, or even more commonly Reishi. There is a bioactive ingredient in the reishi fruiting body which has been found since ancient times in China to be medically active. It is alleged to have several therapeutic effects such as antitumor, anti-inflammatory, anti-bacterial and anti-parasitic effects (Hsieh, Hsu, & Yang, 0; Siew, Chung, & Poh, ). G. lucidum has been used widely in alternative medicine such as naturopathy to treat various human diseases. The cultivation of G. lucidum on substrates such as sawdust or solid pieces of wood has been reported to have been carried out successfully. Lignocellulosic materials such as rubber sawdust are low in protein, and are therefore insufficient for commercially cultivating mushroom. Therefore, the materials require various supplements or additives containing sufficient amount of nitrogen, phosphorus, potassium and vitamins for stimulating the growth and the yield of mushroom. Various supplement for G. lucidum cultivation have been investigated such as wheat bran, rice bran, corn flour, gram flour, rice husks, coconut fiber, peanut hulls, sorghum, sugarcane bagasse, sucrose, molasses, gluten meal and tea waste which were added to the substrate to improve Ganoderma cultivation (Bernabé-González et al., ; Chen, ; Erkel, 0a,b; Gurung, Budhathoki, & Parajuli, ; Paterson-Beedle, Kennedy, Melo, Lloyd, & Medeiros, 0; Peksen & Yakupoglu, 0; Triratana, Thaithatgoon, & Gaugla, ). Especially, the additives, rice bran or wheat bran provide a nitrogen source for enhance growth of primordia of mushrooms (Choi, 0).

4 Page of Songklanakarin Journal of Science and Technology SJST--0.R Seephueak 0 Palm oil sludge (POS) such as decanter cake is a by-product obtained by dehydration of palm oil mill effluent (POME). During the process, most of the solid in the last of the effluent is decanted in a decanter and passed to a filter press before being dried in a rotary drier (Devendra, Yeong, & Ong, ). The composition of nutrients in palm oil sludge contain.00% carbon,.% nitrogen,.% phosphorus,.% potassium, 0.% calcium,.0% protein, 0.% fat,.% fiber and.% ash (Kananam, Suksaroj, & Suksaroj, ). Presently, palm oil sludge is used as fertilizer, soil cover materials in oil palm plantation, bio-gas production (Chavalparit, Rulken, Mol, & Khaodhair, 0; Paepatung, Nophatatana, & Songkasiri, 0) and animal feed (Seephueak, Ngampongsai, & Chanjula, ). Palm oil sludge has a high nutrient content that tends to be used as a supplement in mushroom cultivation. Moreover, use of agro-industrial wastes such as palm oil sludge for mushroom cultivation is good practice in sustainable farming (Oei, 0; Seephueak, Phadungmas, Kaewmano, & Seephueak, ; Seephueak, Preecha, & Seephueak, ). This research aimed to study the efficacy of palm oil sludge as a supplement in mushroom culture (G. lucidum) in plastic bags.. Materials and Methods Culture and their maintenance The culture of G. lucidum was prepared by tissue culture technique in laboratory (Seephueak et al., ). The culture was grown on potato dextrose agar media (PDA) and maintained in slants at 0 C for further studies. Cultivation technique

5 Songklanakarin Journal of Science and Technology SJST--0.R Seephueak Page of 0 Sorghum (Sorghum bicolor) grain spawn was prepared using the standard method (Seephueak et al., ). Palm oil sludge as used as a supplement were collected from palm oil mill belonging to Univanich Palm Oil Public Company Limited, Krabi, Thailand. Samples were sun dried and smashed. The totals of the nutrient in the palm oil sludge and substrates were analyzed at the Scientific Equipment Center, Prince of Songkla University, Hat Yai, Songkhla, Thailand. Nitrogen content was determined by the Micro-Kjeldahal method. Phosphorus content was determined by using a spectrophotometer. Potassium content was determined by flame photometry. Calcium and magnesium content was determined by atomic absorption spectrophotometry (Association of Official Analytical Chemists, ). Rubber sawdust was used as the main substrate for cultivation of G. lucidum, on a dry weight basis mixed with % Ca(OH) and 0.% Mg(SO ) w/w. The substrates were packed in the plastic bags ( g/bag) and the openings of the bag were plugged with cotton and secured with plastic rings. The bags were sterilized by autoclave at C for minutes, and after they had cooled were inoculated with grains of spawn. The inoculated substrate bags were kept at room temperature (- C). After they were fully colonized with mycelia, the bags were exposed in the growth room by opening the top of the plastic bags. Treatment : sawdust + % palm oil sludge Treatment : sawdust + % palm oil sludge Treatment : sawdust + 0% palm oil sludge Treatment : sawdust + % palm oil sludge Treatment : sawdust + % palm oil sludge Treatment : sawdust + % rice bran

6 Page of Songklanakarin Journal of Science and Technology SJST--0.R Seephueak 0 Treatment : sawdust (control) The experimental design The experimental design was a Completely Randomized Design (CRD) with replications. Ten bags were done for each replication. The following data were collected; number of days for full colonization of the mycelia, number of basidiocarp, thickness of pileus, cap diameter and yield. Basidiocarps were harvested when mature ( days/flush). The data were analyzed according to standard method. Means were compared using Duncan s multiple range tests. Biological efficiency (B.E.) was measured using the following formula: Weight of fresh mushroom basidiocarps Biological efficiency or B.E. (%) = 00 Weight of dry substrate. Results and Discussion. Nutrient content in palm oil sludge and substrates It is certain that palm oil sludge has a high nutrient content that can be used as a supplement in mushroom culture, because materials rich in nutrient especially nitrogenous materials give high mushroom yields and fast mycelial growth. Nitrogen, phosphorus and potassium percentages in palm oil sludge were.%, 0.% and 0. %, respectively (Table ). The ratio of carbon to nitrogen (C:N) plays an important role in spawn running. While nitrogen content is also a main factor for developing fruiting bodies on mushroom mycelia. Erkel (0a) reported that the artificial application of a nitrogen source during the mycelial growth stage gave very good results regarding early primordial imitation. Choi (0) suggested that if cotton waste is chosen as the main substrate material for oyster mushroom cultivation, a nitrogen source such as rice bran

7 Songklanakarin Journal of Science and Technology SJST--0.R Seephueak Page of 0 should be added. Bayer and Wilkinson (0) found a correlation between substrate ammonia content and subsequent growth of mushrooms (Alam, Amin, Khair, & Lee, 0; Pokhrel, Kalyan, Budathoki, & Yadav, ). Nitrogen, phosphorus and potassium are chemicals that are important to have in the substrate used for mushroom cultivation. N, P and K content of sawdust supplemented with palm oil sludge varied between 0.%-0.%, 0.0 %-0.0% and 0.%-0.%, respectively and increased with increasing concentration of palm oil sludge in the mixtures. Whereas, using % rice bran the N, P and K content were 0.%, 0.0% and 0.%, respectively. C:N ratio of the substrates decreased when the amount of palm oil sludge increased in the substrate and ranged from. to.. The C:N ratio influenced the time of the beginning of fruiting and the mushroom yield on synthetic media in the case of G. lucidum (Alam et al., 0). In many compost systems, N supplementation can be used to increase mushroom yields by the C:N ratio adjusting between 0: and 0:. In the solid substrate cultivation of G. lucidum, the C:N ratio was a crucial factor for the growth rate of mycelia and also for the formation of fruiting bodies (Alam et al., 0). Calcium content of the substrates varied between.% -.% in palm oil sludge supplement and.% when % rice bran was added. When no supplement was used, the nutrient content was the lowest N, P, K and Ca which were 0.%, 0.0%, 0.% and.% respectively and the C:N ratio was.0. Therefore, this experiment confirmed that palm oil sludge contains nutrients which are suitable for mushroom cultivation, as indicated by previous studies, which using palm oil sludge as a supplement in the same formula for Pleurotus pulmonarius and Auricularia polytricha cultivation (Seephueak et al.,, ).

8 Page of Songklanakarin Journal of Science and Technology SJST--0.R Seephueak 0 Table The nutrient content (%) of the palm oil sludge, sawdust, sawdust supplemented with different percentage of palm oil sludge and % rice bran.. Days of mycelial growth The period of fully colonization of spawn on the substrates had statistically significant differences among the treatments (P<0.0). The periods are shown in Table. In case in which G. lucidum, spawn colonized the substrate of rubber sawdust supplemented with % palm oil sludge had the fastest mycelial growth. The growth period was. days followed by the rubber sawdust supplemented with palm oil sludge %, %, % and 0% were.,.,. and. days, respectively and had no statistical significance when compared with % rice bran was. days. Non-supplemented substrate had the slowest mycelial growth and the period of growth was. days. The results indicated that use of % palm oil sludge supplement in sawdust was the most effective for mycelium development. The average time for full colonization by G. lucidum was. days, while with % rice bran (standard formula), the average time was. days. The results according to a previous study of Seephueak et al. (, ) reported that used of % and % palm oil sludge was a suitable concentration for mycelial growth of phoenix mushroom (P. pulmonarius) and jew s ear (Auricularia polytricha Mont Sacc.) cultivation. The average number of days for full colonization by the mycelia of jew s ear was. days and this was significantly different (P<0.0) from the average by when using % rice bran and non-supplemented sawdust, the number of days for full colonization by mycelia was.0 and. days. The number of days for full colonization of the mycelia of phoenix was.00 days and

9 Songklanakarin Journal of Science and Technology SJST--0.R Seephueak Page of 0 was significantly different (P<0.00) from when using % rice bran and nonsupplemental which this time were. and. days, respectively. Mycelial growth is a preliminary step that creates a suitable condition for fruiting bodies (Pokhrel, Yadav, & Ohga, 0). In experiments, among the seven formulas of substrates for the cultivation of G. lucidum, % palm oil sludge gave the fastest mycelial growth. However, the obtained yields were in reverse order and % palm oil sludge resulted in the highest yield. Liang, Wu, and Wang (0); Noonsong et al. (); Obodai, Cleland-Okine, and Vowotor. (0); Seephueak et al. (, ) reported that the period for spawn to fully colonize the substrate does not correspond with mushroom number and yield. The results indicated that palm oil sludge supplement may have excessive nitrogen and fat contents which had adversely affected the number and yield of the fruiting bodies (Fakoya, Adjumo, & Akinyele, ; Noonsong et al., ). Moreover, Lelley and Janssen () mentioned that higher doses of N rich supplements resulted in temperature increases sufficient to kill the mycelia. According to Heleno et al. (); Regina (0); Rolim, Sales-Compos, Cavalcanti, and Urben () reported that the excess of nitrogen tends to disable the degradation of lignin, retarding, or even inhibiting the mycelial growth, lessening the production of basidiocarp. So that, the nutrient composition of the supplement is one of the most important factors limiting saprophic colonization of cultivated mushrooms and particularly the fruiting of G. lucidum. Table Number of day for full colonization by mycelia of G. lucidum on sawdust supplemented with different percentage of palm oil sludge and % rice bran.

10 Page of Songklanakarin Journal of Science and Technology SJST--0.R Seephueak 0. Number of basidiocarps and yield The number of basidiocarps of G. lucidum on g in a plastic bag of the th flush harvesting are shown in Table and so are the significant differences among the treatments (P<0.0). In the first flush to the rd flush were significances differences among use of supplement and non-use of supplement. While, in the th flush number of basidiocarps were not significantly different. The total of basidiocarp of G. lucidum from the formulas of rubber sawdust supplemented with palm oil sludge were.-.0 basidiocarps/bag which not significantly different from the case of sawdust with % rice bran which was. basidiocarps/bag. Non-supplemented sawdust had the lowest average number of basidiocarps which was. basidiocarps/bag. (Table ).. Diameter of pileus The average diameters of pileus are significantly different (P<0.0) between cases of use of supplement and non-use of supplement. The diameter of pileus was trended to be small between flush. The average diameter of pileus obtained on rubber sawdust supplemented with % palm oil sludge was. cm which was not significantly different from the case of % rice bran sawdust substrate (.0 cm). The smallest diameter of pileus on non-supplemented sawdust was. cm. (Table ).. Thickness of pileus The average thickness of pileus obtained on non-supplement substrate was the highest at.0 cm./basidiocarp, followed by the case of rubber sawdust supplemented with % rice bran at.0 cm./basidiocarp. Using -% palm oil sludge resulted in cm./basidiocarp, not significantly different. (Table ). Table Number of basidiocarps of G. lucidum obtained in the case of sawdust supplemented with different concentrations of palm oil sludge and % rice

11 Songklanakarin Journal of Science and Technology SJST--0.R Seephueak Page 0 of 0 bran Table Diameter of pileus of G. lucidum obtained of sawdust supplemented with different concentrations of palm oil sludge and % rice bran. Table Pileus thickness of G. lucidum obtained from use of sawdust supplemented with different concentrations of palm oil sludge and % rice bran... Yield The highest yield obtained on rubber sawdust supplemented with % palm oil sludge was. g/ bag (B.E.=.0%) followed by %, 0%, % and % of palm oil sludge supplemented sawdust yields. g,. g,. g and.0 g/bag,respectively the biological efficiency were obtained.%,.%,.% and.%, respectively. And this had no significance when compared with % rice bran sawdust where yield was. g/bag and whose biological efficiency was.%. The lowest yield was with the non-supplemented sawdust which was. g/bag (B.E. =.%) (Table ). The highest yield was obtained with substrate supplemented with % palm oil sludge rather than that of the other dosages (-%). The yield of mushroom tended to be decreased with increasing percentages of palm oil sludge. Increasing the dosage of palm oil sludge was negatively correlated with the yield and B.E. According to Erkel (0b), the highest yield was obtained when % molasses than higher dosage. This inverse relationship means that G. lucidum does not need a high dosage of protein and fat for growth. In the experimental, the number of basidiocarps, the average pileus diameter, thickness of pilei and the yield of G. lucidum was the same way. In the first flush G. lucidium gave the high reproduction and trended to be decreased continued when

12 Page of Songklanakarin Journal of Science and Technology SJST--0.R Seephueak 0 nutrient in the substrates decreasing as same as general mushroom cultivation. It clearly shows that using palm oil sludge and rice bran to supplement sawdust were great rather than non-supplemented, significantly different (P<0.0). Yield of G. lucidum varied widely depending on the kind of sawdust and supplement (Erkel, 0b; Gurung et al., ; Rolim et al., ; Roy, Jahan, Das, Munshi, & Noor, ). In the results, % palm oil sludge is the most preferred supplement for use in substrate mixture for G. lucidum cultivation. Average yield determined in the present study to be.0% was higher than those in previous investigations. The yields of G. lucidum on substrates supplemented with rice bran had a biological efficiency of.% (Azizi, Tavana, Farsi, & Oroojalian, ), 0.0% (Gurung et al., ) and.% (Karma & Bhatt, ). Reported yields on wheat bran supplemented sawdust had a biological efficiency of.% (Peksen & Yakupoglu, 0),.% (Karma & Bhatt, ),.% and.% (Roy et al., ) and. % (Gurung et al., ) and on molasses supplemented sawdust was.% (Erkel, 0a). However, yields in this study were lower than when using % tea waste supplemented sawdust had a biological efficiency of.0% (Peksen & Yakupoglu, 0). Because tea waste are high level of nutrition, it could be used alone or in combination with other wastes as a substrate or supplement in A. bisporus (Peker, Baysal, Yigitbasi, Simsek, Colak, & Toker, 0), P. sajor-caju (Dogan & Peksen, 0) and G. lucidum (Yakupoglu, 0; Peksen & Yakupoglu, 0). Whereas, palm oil sludge was suitable used as a supplement only. There are a few reports about use of palm oil mills wastes as a supplement. Petcharat and Thongwiset (0) studied use of oil palm kernel meal as a supplement for A. polytricha and Lentinus squarrosulus. The results showed that % oil palm kernel meal sawdust was suitable for cultivation of A. polytricha (B.E.=.%) and % oil

13 Songklanakarin Journal of Science and Technology SJST--0.R Seephueak Page of 0 palm kernel sawdust was suitable for cultivation of Lentinus squarrosulus (B.E.=. %). Petcharat and Thongwiset (0) studies in abalone mushroom P. cystidiosus the suggested that 0% oil palm kernel meal mixed with sawdust was the optimum concentration for abalone mushroom cultivation (B.E.=.%).While, Seephueak et al. (, ) reported that % and % palm oil sludge in rubber sawdust were suitable for A. polytricha and P. pulmonarius cultivation. However, the use of oil palm kernel as a supplement for mushroom cultivation are more expensive than the use of oil palm sludge which was low prices. This research confirmed that palm oil sludge was the optimum supplement for rubber sawdust for the commercial production of G. lucidum. It could be used as an alternative supplement. Table Yield of G. lucidum obtained from use of sawdust supplemented with different concentrations of palm oil sludge and % rice bran.. Conclusions The effect of palm oil sludge as a supplement on growth and yield of G. lucidum was investigated in this study. % palm oil sludge was suitable concentration for the supplement in G. lucidum cultivation. The mushroom yield on rubber sawdust was. g/bag (B.E.=.0%). Not significantly different (P<0.0) when compared with the % rice bran substrate formula yield which was. g/bag (B.E.=.%). This was a standard formula. Palm oil sludge could be used as an alternative supplement for the commercial production of G. lucidum. As palm oil sludge is the industrial byproducts and is economically cheaper than other supplements, these could have better applicability in low income countries.

14 Page of Songklanakarin Journal of Science and Technology SJST--0.R Seephueak 0 Acknowledgments The authors would like to acknowledge the financial support provided by Rajamangala University of Technology Srivijaya and the Univanich Palm Oil Public Company Limited, Krabi, Thailand for palm oil sludge support. References Alam, N., Amin, R., Khair, A., & Lee, S. (0). Influence of different supplements on the commercial cultivation of Milky white mushroom. Mycobiology, (), -. doi:0./myco.0... Association of Official Analytical Chemists. (). Official method of analysis (th ed). Gaithersburg, Maryland. Azizi, M., Tavana, M., Farsi, M., & Oroojalian, F. (). Yield performance of lingzhi or reishi medicinal mushroom, Ganoderma lucidum (W.Curt.:Fr.) P.Karst. (higher basidiomycetes) using different waste materials as substrates. International Journal of Medicinal Mushrooms, (), -. Bayer, D. M., & Wilkinson, V. L. (0). Spawn, spawning and spawn growth. Mushroom Science and Technology, Pen State Department of Plant Pathology. Bernabé-González, T., Cayetano-Catarino, M., Bernabé-Villanueva, G., Romero-Flores, A., Ángel-Rios, M. D., & Pérez-Salgado, J. (). Cultivation of Ganoderma lucidum on agricultural by products in México. Micologia Aplication International Puebla, (pp. -). Mexico. Chavalparit, O., Rulkens, W. H., Mol, A. P. J., & Khaodhair, S. (0). Options for

15 Songklanakarin Journal of Science and Technology SJST--0.R Seephueak Page of 0 environmental sustainability of the crude palm oil industry in Thailand through enhancement of industrial ecosystems. Environment Development and Sustainability, (), -. Chen, H. M. (). Reutilization of waste materials from a rice distillery for the cultivation of Ganoderma lucidum. (Master thesis, Tunghai University, Taiwan). Choi, K. W. (0). Shelf cultivation of oyster mushroom on the cultivation of an edible fungus (Pleurotus sajor caju; oyster mushroom). (Master thesis, University of Ibadan, Nigeria). Devendra, C., Yeong, S. W., & Ong, H. K. (). The potential of palm oil mill effluent (POME) as a feed source for farms animal in Malaysia. Proceeding in National Workshop on Oil Palm By-Product Utilization. December -. (pp. -). Kuala Lumphur, Malaysia. Dogan, H. & Peksen, A. (0). The effects of substrates prepared by tea waste and disinfection methods on the yield and quality of Pleurotus sajor-caju. Journal Agriculture of Faculty Ondokuz Mayis University,, -. Erkel, E. I. (0a). Yield performance of Ganoderma lucidum (Fr.) Karst cultivation on substrates containing different protein and carbohydrate sources. African Journal of Agricultural Research, (), -. Erkel, E. I. (0b). The effect of different substrate mediums on yield of Ganoderma lucidum (Fr.) Karst. Journal of Food, Agriculture and Environment, (-), -. Fakoya, S., Adejumo, A. F., & Akinyele, J. B. (). Effect of the use of Pycnanthus

16 Page of Songklanakarin Journal of Science and Technology SJST--0.R Seephueak 0 angolensis and different supplements on yields and on the proximate composition of Pleurotus sajor-caju. Journal of Mycology,, -. Gurung, O. K., Budhathoki, U., & Parajuli, G. (). Effect of different substrates on the production of Ganoderma lucidum (Cart.:Fr) Karst. Our Nature, 0, -. Heleno, A. S., Barros, L., Martins, A., Queiroz, M. J., Santos-Buelga, & Ferreira, I.C. (). Fruiting body, spores and invitro produced mycelium of Ganoderma lucidum from Northeast Portugal: A comparative study of the antioxidant potential of phenolic and polysaccharidic extracts. Food Research International Journal,, -. Hsieh, C., Hsu, T. H., & Yang, F. C. (0). Production of polysaccharides of Ganoderma lucidum. Process Biochemistry, (), 0-. Kananam, W., Suksaroj, T. T., & Suksaroj, C. (). Biochemical changes during oil palm (Elaeis guineensis) empty fruit bunches composting with decanter sludge and chicken manure. Science Asia,, -. doi: 0./scienceasia Karma, A., & Bhatt, A. B. (). First attempt of an organic cultivation of red Ganoderma lucidum subtropical habitat and its economics. International Journal of Pharmacy and Pharmaceutical Sciences, (), -. Liang, Z. C., Wu, C. Y., & Wang, J. C. (0). The evaluation of using mushroom sawdust wastes for cultivation of Pleurotus citrinopileatus. Fungal Science, (-), -. Lelley, J. I., & Janssen, A. (). Productivity improvement of oyster mushroom

17 Songklanakarin Journal of Science and Technology SJST--0.R Seephueak Page of 0 substrate with a controlled release of nutrient. Mushroom News,, -. Noonsong, V., Puttakun, N., Tinsirisuk, M., & Seephueak, P. (). Recycling of spent Pleurotus compost for production of the Agrocybe cylindracea. Mycosphere, (), -. Obodai, M., Cleland-Okine, J., & Vowotor, K. A. (0). Comparative study on the growth and yield of Pleurotus ostreatus mushroom on different lignocellulosic by-products. Journal of Industrial Microbiology and Biotechnology, (), -. Oei, P. (0). Small scale mushroom cultivation. Agrodok. Wageningen, The Netherlands, CTA publishing. Paepatung, N., Nophatatana, N., & Songkasiri, W. (0). Biomethane potential of biological solid and agricultural wastes. Asian Journal on Energy and Environment, 0(), -. Paterrson-Beedle, M., Kennedy, J. Melo, F. A. D., Lloyd, L. L., & Medeiros, V. (0). A cellulosic expolysaccharide produced from sugarcane molasses by a Zoogloea sp. Carbohydrate and Polymers Journal, (), -. Peker, H., Baysal, E., Yigitbasi, O. N., Simsek, H., Colak, M., & Toker, H. (0). Cultivation of Agaricus bisporus on wheat straw and waste tea leaves based compost formulas using wheat chaff as activator material. African Journal of Biotechnology,, 0-. Peksen, A., & Yakupoglu, G. (0). Tea waste as a supplement for cultivation of Ganoderma lucidum. World Journal Microbiology and Biotechnology, (), -.

18 Page of Songklanakarin Journal of Science and Technology SJST--0.R Seephueak 0 Petcharat, V., & Tongwised, A. (0). Effect of oil palm kernel meal on yield of Hed Hu Hnu (Auricularia polytricha (Mont.) Sacc.) and Hed Khon Khoa (Lentinus squarrosulus Mont.). Sonklanakaria Journal Science and Technology, (), -. Petcharat, V., & Tongwised, A. (0). Use of oil palm kernel meal as a supplement material for abalone mushroom (Pleurotus cystidiosus O.K. Miller) cultivation. Songklanakarin Journal Science and Technology, (), -. Pokhrel, C. P., Yadav, R. K. P., & Ohga, S. (0). Effects of physical factors and synthetic media on mycelial growth of Lyophyllum decastes. Journal of Ecobiotechnology, (), -. Pokhrel, C. P, Kalyan N., Budathoki U., & Yadav, R. K. P. (). Cultivation of Pleurotus sajor-caju using different agricultural residues. International Journal of Agricultural Policy and Research, (), -. Regina, M. (0). Cinética do crescimento miceliano de Lentinula edodes (Berk.) Peglerem bagaço de cana-de-açúcar e serragem de eucalipto. (Master thesis, Universidade Estadual Paulista, Botucatu, Brazil). Rolim, L. N., Sales-Campos, C., Cavalcanti, M. A. Q., & Urben, A.F. (). Application of chinese Jun-Cao technique for the production of Brazilian Ganoderma lucidum strains. Brazilian Archives of Biology and Technology, (), -. Roy, S., Jahan, M. A. A., Das, K. K., Munshi, S. K., & Noor, R. (). Artificial cultivation of Ganoderma lucidum (Reishi Medicinal Mushroom) using different sawdusts as substrates. American Journal of Bioscience, (), -.

19 Songklanakarin Journal of Science and Technology SJST--0.R Seephueak Page of 0 Seephueak, P., Phadungmas, P., Kaewmano, P., & Seephueak, W. (). Use of palm oil sludge as a supplement material for phoenix mushroom (Pleurotus pulmonarius) cultivation. Khon Kaen Agriculture Journal (Supplement), (), -. Seephueak, P., Preecha, C., & Seephueak, W. (). Effect of nutrient in palm oil sludge on mycelium growth of Auricularia polytricha (Mont.) Sacc. Khon Kaen Agriculture Journal (Supplement), (), -. Seephueak, W., Ngampongsai, W., & Chanjula, P. (). Effects of palm oil sludge in concentrate on nutrient utilization and rumen ecology of Thai native cattle fed with hay. Songklanakarin Journal Science and Technology, (), -0. Siew, K. C., Chung, L. L., & Poh, G. C. (). Effect of drying on crude Ganoderic acids and water soluble polysaccharides content in Ganoderma lucidum. International Journal of Pharmacy and Pharmaceutical Sciences, (), -. Triratana, S., Thaithatgoon, S., & Gaugla, M. (). Cultivation of Ganoderma lucidum in sawdust bags. The International Society of Mushroom Science,, -. Yakupoglu, G. (0). The effects of different substrates on yield and quality of Ganoderma lucidum mushroom. (Master thesis, Onokuz Mayis University, Turkey).

20 Page of Songklanakarin Journal of Science and Technology SJST--0.R Seephueak 0 Table The nutrient content (%) of the palm oil sludge, sawdust, sawdust supplemented with different percentage of palm oil sludge and % rice bran. Nutrient content (%) Substrate C N P K Ca C/N ratio. palm oil sludge sawdust sawdust + % palm oil sludge sawdust + % palm oil sludge sawdust + 0% palm oil sludge sawdust + % palm oil sludge sawdust + % palm oil sludge sawdust + % rice bran Source: Seephueak et al. ()

21 Songklanakarin Journal of Science and Technology SJST--0.R Seephueak Page of 0 Table Number of day for full colonization by mycelia of G. lucidum on sawdust supplemented with different percentage of palm oil sludge and % rice bran. No. of day for full colonization of Substrate ( g/bag) mycelia. sawdust + % palm oil sludge. ±.0 cd. sawdust + % palm oil sludge. ±. cd. sawdust + 0% palm oil sludge. ±. bc. sawdust + % palm oil sludge. ± 0. d. sawdust + % palm oil sludge. ± 0. cd. sawdust + % rice bran. ±.00 cd. sawdust (control). ±. a CV (%). Significance difference ** Means within a column followed by same letter are not significantly different (P<0.0) by DMRT.

22 Page of Songklanakarin Journal of Science and Technology SJST--0.R Seephueak 0 Table Number of basidiocarps of G. lucidum obtained in the case of sawdust supplemented with different concentrations of palm oil sludge and % rice bran. No. of basidiocarp/bag Substrate ( g/bag) Total st flush nd flush rd flush th flush. sawdust + % palm oil sludge. a. a.00 a.00. a. sawdust + % palm oil sludge. a. a.0 a.00.0 a. sawdust + 0% palm oil sludge. a. a.00 a.0. a. sawdust + % palm oil sludge. a. a.00 a 0.. a. sawdust + % palm oil sludge. a. a.00 a 0.. a. sawdust + % rice bran.0 a. a.0 a.0. a. sawdust (control) 0.b 0. b 0. b -. b CV (%)..... Significant difference ** ** ** ns ** Means within a column followed by same letter are not significantly different (P<0.0) by DMRT. - No fruiting body

23 Songklanakarin Journal of Science and Technology SJST--0.R Seephueak Page of 0 Table Diameter of pileus of G. lucidum obtained of sawdust supplemented with different concentrations of palm oil sludge and % rice bran. Diameter of pileus (cm/basidiocarp) Substrate ( g/bag) st flush nd flush rd flush th flush Aver.. sawdust + % palm oil sludge. a. a. a.. a. sawdust + % palm oil sludge. sawdust + 0% palm oil sludge. sawdust + % palm oil sludge. a. a. a.. a. sawdust + % palm oil sludge. sawdust + % rice bran. sawdust (control). a. a. a.. a. a. a. ab.. a. a.0 a. a.. a. a. a. a..0 a. b.0 b. b -. b CV (%) Significant Difference ** ** ** ns ** Means within a column followed by same letter are not significantly different (P<0.0) by DMRT. - No fruiting body

24 Page of Songklanakarin Journal of Science and Technology SJST--0.R Seephueak 0 Table Pileus thickness of G. lucidum obtained from use of sawdust supplemented with different concentrations of palm oil sludge and % rice bran. Thick of pileus (mm/bag) Substrate ( g/bag) st flush nd flush rd flush th flush Aver.. sawdust + % palm oil sludge b sawdust + % palm oil sludge. sawdust + 0 % palm oil sludge. sawdust + % palm oil sludge b sawdust + % palm oil sludge. sawdust + % rice bran. sawdust (control) 0..0 ab ab b b a CV (%)..... Significant Difference ns ** ns ns ns Means within a column followed by same letter are not significantly different (P<0.0) by DMRT. - No fruiting body

25 Songklanakarin Journal of Science and Technology SJST--0.R Seephueak Page of 0 Table Yield of G. lucidum obtained from use of sawdust supplemented with different concentrations of palm oil sludge and % rice bran. Yield (g/bag) Substrate ( g/bag) st nd rd th flush flush flush flush Total B.E. (%). sawdust + % palm oil sludge.. a. ab.. a.0 a. sawdust + % palm oil sludge.. a. a.. a. a. sawdust + 0% palm oil sludge.. a. abc.. a. a. sawdust + % palm oil sludge.00. a. a.. a. a. sawdust + % palm oil sludge..0 a. bc.00.0 a. a. sawdust + % rice bran..0 a. ab.. a. a. sawdust (control).. b. c -. b. b CV (%) Significance difference ns ** ** ns ** ** Means within a column followed by same letter are not significantly different (P<0.0) by DMRT. - No fruiting body