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Cultivation of Flammulina Velutipes (Golden Needle Mushroom/Enokitake) on Various Agroresidues

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Cultivation of Flammulina Velutipes (Golden Needle Mushroom/Enokitake) on Various Agroresidues CULTIVATION OF FLAMMULINA VELUTIPES (GOLDEN NEEDLE MUSHROOM/ENOKITAKE) ON VARIOUS AGRORESIDUES NOORAISHAH BINTI HARITH FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR 2014 CULTIVATION OF FLAMMULINA VELUTIPES (GOLDEN NEEDLE MUSHROOM/ENOKITAKE) ON VARIOUS AGRORESIDUES NOORAISHAH BINTI HARITH DISSERTATION SUBMITTED IN FULLFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE INSTITUTE OF BIOLOGICAL SCIENCES FACULTY OF SCIENCE UNIVERSITY OF MALAYA KUALA LUMPUR 2014 iii ABSTRACT Sawdust and rice bran are common commercially used fruiting substrate components for the cultivation of Flammulina velutipes, or known as ‘golden needle mushroom’ in Malaysia. Due to the declining of sawdust supply, and the abundance of lignocellulosic agroresidues in Malaysia, hence, this study was carried out to investigate the possibility of using palm oil wastes; such as empty fruit bunches (EFB), palm pressed fiber (PPF), and paddy straw (PS) from rice plantation, as base carbon-sources in fruiting substrate used as either singular or in combination with different agroresidues. The percentage of rice bran (RB) and spent yeast (SY) used as the nitrogen-sources supplemented were also investigated. Mycelium growth and density, yield of mushroom and biological efficiency (BE) were the parameters determined to evaluate singular and different combination of substrates tested. For the improvement of F. velutipes inoculum addition of growth hormone used in this study consisting of β- indole acetic acid (IAA) combined with 6-benzylaminopurine (BAP) at a concentration of 0.5 mg/L each enhanced mycelial growth rate at 10.53 mm/day compared to non- supplemented malt extract agar (MEA) media (7.83 mm/day). All the agro-residues tested showed good potential to be used as fruiting substrates for the cultivation of F. velutipes based on mycelial and basidiocarp yield. For singular substrate, PPF (100) and EFB (100) showed higher mean radial growth rates of mycelium of 6.64 and 6.17 mm/day respectively, compared to other agroresidues. Among the formulations, combination of substrates, SD+PPF (75:25), PS+PPF (50:50) and SD+PS (50:50) showed higher mycelial growth rates of 7.20, 6.84 and 6.78 mm/day respectively. In terms of basidiocarp yield, EFB+PS (75:25), PS+PPF (50:50), and PPF (100) gave highest BE of 185.09, 150.89, and 129.06% respectively. Nitrogen supplementation with rice bran and spent yeast at levels of 5.0 – 20.0% concentrations showed no significant effects based on mycelial growth rate, basidiocarp yield and BE. These ii fruiting substrate formulations would be a good alternative for the growers of F. velutipes since they are easily available in abundance and low-cost. iii ABSTRAK Habuk kayu dan beras lazim digunakan secara komersil sebagai substrat penjanaan buah untuk penanaman Flammulina velutipes, yang dikenali sebagai 'cendawan jarum emas' di Malaysia. Disebabkan oleh pengurangan bekalan habuk kayu, dan sisa-agro lignoselulosa yang banyak didapati di Malaysia, kajian ini dijalankan untuk mengkaji kemungkinan penggunaan bahan buangan kelapa sawit; seperti tandan buah kosong (EFB) dan serat ditekan sawit (PPF), dan jerami padi (PS) dari tanaman padi, sebagai sumber asas karbon substrat penjanaan buah sama ada dalam bentuk tunggal atau dalam kombinasi dengan sisa-agro berbeza. Peratus dedak beras (RB) dan yis terpakai (SY) yang digunakan sebagai sumber nitrogen tambahan juga dikaji. Pertumbuhan dan ketebalan miselium, penghasilan cendawan dan efisiensi biologi (BE) adalah parameter yang diperolehi untuk menilai keberkesanan penggunaan substrat tunggal dan gabungan substrat yang diuji. Untuk penambahbaik strain F. velutipes, kajian ini juga mengkaji kesan hormon pertumbuhan terhadap pertumbuhan miseliumyang digunakan dalam penyediaan inokulum benih. Hormon pertumbuhan yang digunakan dalam kajian ini ialah β-indole asid asetik (IAA) dan 6-benzil amino purina (BAP). Kombinasi kepekatan 0.5 mg/L BAP+0.5 mg/L IAA menunjukkan kadar pertumbuhan miselium yang tertinggi dengan nilai 10.53 mm/hari, manakala kadar pertumbuhan miselia pada MEA tanpa penambahan hormon adalah 7.83 mm/hari. Semua sisa-agro yang diuji menunjukkan keupayaan positif untuk digunakan sebagai substrat janabuah, berdasarkan keupayaan pertumbuhan miselium dan penghasilan janabuah. Bagi substrat tunggal, PPF (100) dan EFB (100) menunjukkan purata kadar pertumbuhan miselia secara radial yang tertinggi, 6.64 dan 6.17 mm/hari mengikut turutan, berbanding dengan sisa-sisa pertanian lain. Kombinasi substrat, SD+PPF (75:25), PS+PPF (50:50) dan SD+PS (50:50) menunjukkan kadar pertumbuhan miselia yang tinggi, 7.20, 6.84 dan 6.78 mm/hari mengikut turutan. Dalam penghasilan iv janabuah, EFB+PS (75:25), PS+PPF (50:50), dan PPF (100) mencatatkan BE yang tertinggi, 185.09, 150.89, dan 129.06% mengikut turutan. Tiada kesan ketara pada kadar pertumbuhan miselia dan BE. Formulasi substrate ini merupakan alternatif yang baik bagi penanam cendawan F. velutipes kerana ia merupakan lignoselulosa sisa-agro yang mudah diperolehi dengan banyak dan kos yang rendah . v ACKNOWLEDGEMENTS In the name of Allah, the most beneficent and the most merciful. First and above all, all praise to Allah, the almighty for providing me this opportunity and granting me the capability to complete this thesis successfully. This thesis appears in its current form due to the assistance and guidance of several people. I would therefore like to offer my sincere thanks to all of them. My first utmost gratitude goes to my supervisor, Prof. Dr. Noorlidah binti Abdullah for accepting me as a master student, her warm encouragement, thoughtful guidance, critical comments, patience and support in every stage of this study. I would like to express my deep thanks to my co-supervisor, Prof. Dr. Vikineswary Sabaratnam for offering valuable advice, support, and especially for her patience during the whole period of this study. My greatest appreciation goes to all my friends in the Mycology Laboratory and Fungal Biotechnology Laboratory for their help and support during my struggles and frustrations since the friendship were bond. I would like to express my heartfelt gratitude to my family, especially my mother, Joanna Joy binti Abdullah, and brother, Mohamed Johari bin Harith, for always believing in me, for their continuous love, prayer and support in my decisions. Without whom I could not have made it here. Deeply thanks to my supportive friend, Noor Afzan binti Rosli, who will always be a true friend of mine. Thank you. Nooraishah Harith vi TABLE OF CONTENTS ABSTRACT ii ABSTRAK iv ACKNOWLEDGEMENTS vi TABLE OF CONTENTS vii LIST OF FIGURES x LIST OF TABLES xii LIST OF SYMBOLS AND ABBREVIATIONS xiv CHAPTER 1.0 INTRODUCTION 1 CHAPTER 2.0 LITERATURE REVIEW 4 2.1 Mushroom Cultivation 4 2.1.1 Inoculum (spawn) production 6 2.1.2 Fruiting substrate formulation 10 2.1.3 Mushroom growing 13 2.2 Flammulina velutipes (Curtis) Singer 13 2.2.1 Morphology 14 2.2.2 Nutritional value and medicinal properties of F. velutipes 16 2.2.3 Environmental factors affecting fruiting of F. velutipes 16 2.3 Agricultural Lignocellulosic Wastes in Malaysia 19 2.3.1 Agroresidues derived from rice cultivation 21 2.3.2 Agroresidues derived from palm oil mill 23 2.3.3 Brewery solid waste 24 vii CHAPTER 3.0 MATERIALS AND METHODS 25 3.1 Flammulina velutipes Culture 25 3.2 Effect of Plant Growth Hormones on Mycelial Growth of F. velutipes on 25 Malt Extract Agar (MEA) 3.2.1 Preparation of mycelium culture and measurement of growth 25 3.2.2 Experimental design to determine the effect of hormone on mycelial 25 growth 3.3 Selection of Various Lignocellulosic Agroresidues as The Base Carbon- 29 source for Fruiting Substrate of F. velutipes 3.3.1 Preparation of alginate immobilized mycelium of F. velutipes as 29 inoculum 3.3.2 Selection of fruiting substrates 29 3.4 Effect of Different Levels of Nitrogen-source Supplementation on Selected 33 Substrate Formulations on Yield of F. velutipes 3.4.1 Preparation of fruiting substrates supplemented with nitrogen-source 33 3.4.2 Experimental design for nitrogen supplementation 34 3.5 Statistical Analysis 35 CHAPTER 4.0 RESULTS 36 4.1 Effect of Growth Hormones on Mycelial Growth of F. velutipes for The 36 Preparation of Spawn 4.1.1 Optimisation of hormone concentrations 40 4.1.2 Verification 45 4.2 Selection of Carbon-source consisting of Agroresidues used in Fruiting 45 Substrate Formulation for F. velutipes Cultivation 4.3 Effect of Supplementation of Nitrogen-source on Mycelial Growth and Yield 51 of F. velutipes 4.3.1 Analysis of effect nitrogen-source supplementation for PS+EFB (25:75) 54 viii as main carbon-source 4.3.2 Analysis of effect nitrogen-source supplementation for PS+PPF (50:50) 58 as main carbon-source 4.3.3 Analysis of effect nitrogen-source supplementation for PPF (100) as 61 main carbon-source CHAPTER 5.0 DISCUSSION 64 5.1 Effect of Growth Hormones on F. velutipes Mycelial Growth 64 5.2 Selection of Agroresidues as Carbon-source in The Formulations of Fruiting 66 Substrate for F. velutipes 5.3 Effect of Rice Bran and Spent Yeast as Supplementaion of Nitrogen-sources 73 for Mycelial Growth and Yield of F. velutipes CHAPTER 6.0 CONCLUSION 76 REFERENCES 78 APPENDICES 93 Appendix A 93 Appendix B: Chemical Composition 94 Appendix C: Experimental Data 95 Appendix D: Statistical Analysis 106 Appendix E: Publications 115 ix LIST OF FIGURES Figure 2.1 Wild F. velutipes 15 Figure 2.2 Cultivated F. velutipes 15 Figure 3.1 Carbon-source substrates: SD (sawdust), PS (paddy straw), 30 EFB (empty fruit bunches), and PPF (palm pressed fiber). Figure 3.2 Nitrogen-source supplements used: rice bran (RB) and spent 33 yeast (SY) Figure 4.1 Residual plot for F. velutipes supplemented with IAA and 39 BAP Figure 4.2 Pareto chart of standardized effects for F. velutipes 40 supplemented with IAA and BAP Figure 4.3 Main effects plot (data means) for mycelia growth rate of F.
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