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.
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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 .
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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
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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
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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. 40 velutipes supplemented with IAA and BAP
Figure 4.4 Residual plot for F. velutipes supplemented with IAA and 43 BAP
Figure 4.5 Contour plot of mycelia growth rate versus plant growth 44 hormones
Figure 4.6 Surface plot of mycelia growth rate versus plant growth 44 hormones
Figure 4.7 Mycelia thickness (From left; sparse, and dense) 50
Figure 4.8 Primordia formation on the surface at the top of a fruiting bag 50
Figure 4.9 Fresh F. velutipes basidiocarps after harvest 51
Figure 4.10 Residual plots for mycelial growth rate (mm/day) of F. 56 velutipes on PS+EFB (25:75) supplemented with different concentrations of RB and SY.
Figure 4.11 Pareto chart of standardized effects for mycelia growth rate 57 (mm/day) of F. velutipes on PS+EFB (25:75) supplemented with different concentrations RB and SY.
Figure 4.12 Main effects plot (data means) for mycelial growth rate 57 (mm/day) of F. velutipes on PS+PPF (25:75) supplemented with different concentrations RB and SY
x
Figure 4.13 Residual plots for mycelial growth rate (mm/day) of F. 59 velutipes on PS+PPF (50:50) supplemented with different concentrations of RB and SY.
Figure 4.14 Pareto chart of standardized effects for mycelial growth rate 60 (mm/day) of F. velutipes on PS+PPF (50:50) supplemented with different concentrations RB and SY.
Figure 4.15 Main effects plot (data means) for mycelial growth rate 61 (mm/day) of F. velutipes on PS+PPF (50:50) supplemented with different concentrations RB and SY.
Figure 4.16 Residual plots for mycelia growth rate (mm/day) of F. 63 velutipes on PPF (100) supplemented with different concentrations of RB and SY.
Figure 4.17 Pareto chart of standardized effects for mycelia growth rate 63 (mm/day) of F. velutipes on PPF (100) supplemented with different concentrations RB and SY
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LIST OF TABLES
Table 3.1 Experimental factors and levels for screening 26
Table 3.2 The experimental design of various combination 27 concentrations of plant growth hormones for screening
Table 3.3 Experimental factors and levels for optimization 27
Table 3.4 The experimental design of various combination 28 concentrations of plant growth hormones for optimization
Table 3.5 The percentage of carbon and nitrogen in lignocellulosic by- 30 products used as fruiting substrates
Table 3.6 The percentage of carbon and nitrogen in rice bran (RB) and 33 spent yeast (SY)
Table 3.7 Experimental factors and levels 34
Table 3.8 The experimental design of various combination 35 concentrations of nitrogen-source substrates
Table 4.1 Growth rate of F. velutipes mycelium grown on MEA 36 supplemented with different plant growth hormones concentrations
Table 4.2 Estimated effects of growth hormones, coefficients, T-value 37 and P-value for mycelial growth rate (mm/day)
Table 4.3 Analysis of variance (ANOVA) for mycelial growth rate 38 (mm/day) on the supplemented MEA media with growth hormones
Table 4.4 Optimization of growth hormone concentration (mg/L) on 41 the growth rate of F. velutipes mycelium (mm/day)
Table 4.5 Estimated regression coefficients, T-value and P-value for 42 mycelia growth rate (mm/day).
Table 4.6 Analysis of variance (ANOVA) for mycelia growth rate 42 (mm/day) on the supplemented MEA media with growth hormones.
Table 4.7 Predicted value of mycelial growth rate (mm/day) at 45 optimum concentration of growth hormones
Table 4.8 Effect of various carbon-source agroresidues on the radial 47 mycelia growth rate of F. velutipes (mm/day).
Table 4.9 Effect of selected fruiting substrate formulations on the 49 mycelia growth rate (mm/day), mycelium thickness, yield of xii
F. velutipes basidiocarp (g) and biological efficiency (%).
Table 4.10 Effect of nitrogen supplements on the average mycelial 53 growth rate (mm/day), basidiocarp yield (g) and biological efficiency (%) of F. velutipes
Table 4.11 PS+EFB (25:75): Estimated effects of nitrogen-sources 55 supplementation, coefficients, t-value and p-value for mycelial growth rate (mm/day).
Table 4.12 PS+EFB (25:75): Analysis of variance (ANOVA) for 55 mycelial growth rate (mm/day) on supplementation substrates
Table 4.13 PS+PPF (50:50): Estimated effects of nitrogen-sources 58 supplementation, coefficients, t-value and p-value for mycelial growth rate (mm/day).
Table 4.14 PS+PPF (50:50): Analysis of variance (ANOVA) for 59 mycelial growth rate (mm/day) on supplementation substrates.
Table 4.15 PPF (100): Estimated effects of nitrogen-sources 62 supplementation, coefficients, t-value and p-value for mycelial growth rate (mm/day).
Table 4.16 PPF (100): Analysis of variance (ANOVA) for mycelial 62 growth rate (mm/day) on supplemented substrates.
xiii
LIST OF SYMBOLS AND ABBREVIATIONS
% percentage
˚C degree Celcius
B.E. biological efficiency
BAP 6-benzylaminopurine
C carbon
C:N carbon to nitrogen
CaCO3 calcium carbonate
CCD central composite design cm centimetre
EFB empty fruit bunches g gram g/L gram per litre
IAA indole-3-acetic acid kg kilogram
M molar
ME malt extract
MEA malt extract agar mg miligram mg/L miligram per litre mm milimetre mm/day milimetre per day mm/day milimetre per day
MT metric ton
N nitrogen
NaOH sodium hydroxide
xiv
PPF palm pressed fibres ppm parts per million
PS paddy straw psi pounds per square inch
RB rice bran
RM Malaysian ringgit
RSM response surface method
SD Sawdust sp. Singular for species spp. Plural for species
SSF solid state fermentation
SY brewery's spent yeast
xv
CHAPTER 1.0 INTRODUCTION
Basidiomycetes mushroom is the premier recycler on the planet and produces fruiting bodies called basidiocarps. In Malaysia, mushroom cultivation is a promising industry, with many new businesses developing every year. Most of the mushrooms in the Malaysian market are imported from China. The import of mushrooms in Malaysia increases every year. According to Datuk Seri Ismail Sabri Yaakob, the Minister of
Agriculture and Agro-Based Industry of Malaysia, in the year of 2013, imported mushrooms are 84%, while Malaysia produced mushroom are 16% (Siti Suraya Md
Top, 2014). Flammulina velutipes (Curtis) Singer, or simply known as ‘cendawan jarum emas’ (Malaysia) or ‘golden needle mushroom’ and ‘enokitake’ (Japan), is one of the major imported mushroom in Malaysia. It is imported from Taiwan, China and South
Korea. In 1997, F. velutipes ranked fifth in total worldwide production of edible mushrooms with China, Japan, South Korea and Taiwan as the leading producers (Kües and Liu, 2000). The mushroom is available either fresh or canned, but the fresh mushroom is preferred for consumption.
The white basidiocarps with tiny caps and long stems of F. velutipes contains 17
- 31% crude protein, 1.9 - 5.8% fat, 3.7% fiber and 7.4% ash (Stamets, 2000). These nutritional properties not only make the mushroom a very good dietary food, but also have positive effect on human health. Polysaccharides from F. velutipes have been shown to strongly stimulate host mediated antitumor responses. A new immuno- modulating protein that stimulates the production of human peripheral blood lymphocytes was isolated by Ko et al. (1995) in Japan. Dr. Ikekawa of the national cancer institute of Tokyo conducted an epidemiological survey of enokitake growers in
1989 and found that their families had substantially lower cancer rates than the average cancer rates in Japan or that of their surrounding community in Nagano, Japan. This species also produces a target specific antibiotic, which may be significant in the 1 development of future antibiotics. Flammulina velutipes possess antitumor (Ikekawa et al., 1982), antioxidants (Bao et al., 2009; 2008), and cholesterol-lowering activities
(Fukushima et al., 2001). It can also prevent high blood pressure, and used for the treatment of liver disease, and care for gastric ulcer (Chang and Miles, 2004).
Naturally, F. velutipes is a short, furry-footed mushroom that grows on dead trunks, stumps of broad-leaves, and rarely on dead stumps of conifer. In temperate zone countries, rural people collect the mushroom as a food source from late autumn to spring. Flammulina velutipes is initially cultivated by using wood logs, but the quality of the mushrooms was inferior. Now, cultivation on sawdust is commonly employed since white, stiff, and durable sporocarps are preferred. The mushroom is cultivated for
30 days in a plastic bottle or a vinyl bag at 15°C and 70% humidity (Tonomura, 1978).
In Malaysia, the production of F. velutipes by growers is still insufficient and inefficient. There are two major obstacles faced by our local mushroom growers.
Firstly, the limited supply of sawdust mostly due to the competition from other industries. Second, sawdust supplies are often mixed with chemicals used in the processing industry. The tainted supply of sawdust affected mushroom growth – low yield, high percentage of contamination and unsynchronized flushing patterns.
Therefore, it is imperative that other sources of substrates be utilized for mushroom cultivation. In Malaysia, large volumes of unused lignocellulosic agroresidues can be found. These agroresidues are left to rot in the field or are disposed of through burning.
Cultivation of mushrooms on these agroresidues may be a solution to transforming these inedible wastes into accepted edible biomass of high market value.
Hence, based on the reasons stated above, the objectives of this project were as to:
i) investigate the effect of plant growth hormones on growth of F. velutipes
mycelium for the preparation of spawn
2 ii) select lignocellulosic agroresidues as fruiting substrates for F. velutipes
cultivation iii) investigate the effect of supplementation of nitrogen-sources on basidiocarp
yield.
3
CHAPTER 2.0 LITERATURE REVIEW
2.1 Mushroom Cultivation
Mushroom cultivation has proved to be an economically and ecologically important biotechnology industry for efficient utilization, value-added and biotransformation of agroindustrial residues to produce value-added products (Chang
2001, Chiu et al. 2000, Zervakis and Philippoussis, 2000). This industry has been expanded all over the world in the past few decades. According to Kües and Liu (2000), the worldwide production of commercial mushroom (basidiocarps) comprises about 5 x
106 MT fresh weight year-1, although only a few basidiomycetes (Agaricus, Lentinula,
Pleurotus, Auricularia, Volvariella, Flammulina, Tremella, and Ganoderma) can be cultivated. Flammulina velutipes was at fifth rank of total worldwide production of edible mushrooms in 1997, totaled 280,000 MT with China, Japan, South Korea and
Taiwan as the leading producers (Chang, 1999). Historically, F. velutipes cultivation started during the 8th century in China (Wang, 1995; Yang, 1986). Initially, the cultivation was done on wood logs, however in 1928, it was first cultivated on sawdust and rice bran in Japan (Nakamura, 1981). Now, cultivation on sawdust is the method commercially used in China, Japan, South Korea and Taiwan.
Commercial mushroom cultivation, carried out either in large or small scale, is an efficient solid-state fermentation (SSF) process of food protein recovery from lignocellulosic agroresidues by the degradation capabilities of mushroom (Chiu and
Moore, 2001; Martínez-Carrera et al., 2000). Zhang (2008) reported that more than 200 x 109 tons per year of lignocellulosic biomass have been produced on the surface of
Earth. The majority of this organic matter is not directly edible by humans and animals, and it causes environmental pollution problems (Laufenberg et al., 2003). The chemical properties of those lignocellulosic materials can be converted by SSF into various different value-added products including mushrooms, animal feed enriched with
4 microbial biomass, compost to be used as biofertilizer or biopesticide (Zervakis et al.,
2005), enzymes (Howard et al., 2003), organic acids (Pandey et al., 2000), ethanol (Kim and Dale, 2004), flavours (Sánchez et al.,2002, Manpreet et al., 2005), biologically active secondary metabolites (Manpreet et al. 2005), bioremediation of hazardous compounds (Tengerdy and Szakacs, 2003), biological detoxification of agroindustrial residues (Tengerdy and Szakacs, 2003), and bio-pulping (Krishna 2005, Nigam et al.,
2004)
Cultivation of F. velutipes utilized formulated substrate contained in polypropylene bottles or bags. Besides sawdust, the substrates most utilized are agricultural residues, such as corncobs, cottonseed husk, sugarcane bagasse, etc.,
(Wang, 1995; Royse, 1995; Fan et al., 1990; Chang, 1989; Yang, 1986). Other higher basidiomycetes mushrooms species such as Lentinula edodes (Berk.) Pegler and
Pleurotus spp. reveal high efficiency in degradation of a wide range of lignocellulosic residues, such as wheat straw, cotton wastes, coffee pulp, corn cobs, sunflower seed hulls wood chips and sawdust, peanut shells, vine prunings and others into mushroom protein (Philippoussis et al., 2000, 2001; Stamets 2000; Ragunathan et al. 1996,). The productivity of the conversion is being expressed by biological efficiency (BE) (Chang et al. 1981). The mushroom mycelium produces significant quantities of a plethora of enzymes, which can degrade lignocellulosic agroresidues and use them as nutrients for their growth and fructification (Elisashvili et al. 2008; Bushwell et al. 1996). The nature and nutrient composition of the substrate affect mycelial growth, mushroom quality and yield in this value-added biotransformation process (Philippoussis et al. 2001, 2003;
Kües and Liu 2000).
There are three major stages involved in mushroom cultivation: (1) inoculum
(spawn) production, (2) substrate preparation, and (3) mushroom growing i.e. inoculation of the substrate with spawn of mushroom mycelia, growth of mycelia to 5 colonize the substrate, induction of fruiting, harvesting, and processing of the basidiocarps (Martínez-Carrera et al., 2000; Wang, 1999).
2.1.1 Inoculum (spawn) production
The mushroom “seed” (propagation material) is generally referred as spawn. In order to achieve reliable and vigorous fungal growth and basidiocarp production of good quality, high growth rate of mycelial inoculum is necessary. The selection and breeding work is an essential prerequisite to acquire suitable mycelial culture for commercial cultivation, which ensures good yield and quality (Wood, 1989). Spawn- making is a rather complex task, not feasible for the common mushroom grower, and is produced by specialist companies (spawn-makers) using large scale bulk autoclaving, clean air and other microbiological sterile techniques for the growth of vegetative mycelium onto solid substrate as nutrients such as cereal grains, wood chips and sawdust (Mata and Savoie, 2005; Stamets, 2000).
The colonized mixture of cereal grain and mycelium is called spawn and is grown under axenic conditions in either autoclavable polyethylene bags or jars, as to ensure gas exchange to occur. Moisture content also plays a critical role in the successful colonization by mushroom mycelium of sterilized grain (Stamets, 2000). If the grain is too dry, growth is retarded, with the mycelium forming fine threads and growing slowly. If too much water is added, the grain clumps and dense, slow growth occurs. Higher moisture contents also encourage bacterial blooms. Without proper moisture content, spawn production is hampered, even though all other techniques may be perfect.
Finally, after quality control to assure biological purity and vigor, spawn is distributed from the manufacturer to individual mushroom farms in the same aseptic containers used for spawn production (Royse, 2002; Wood and Smith, 1987).
6
2.1.1.1 The effect of plant growth hormones on mycelial growth of mushroom
Plant hormones are involved in several stages of plant growth and development.
Previous studies had investigated the effect of hormones on the growth of bacteria and fungi. Mukhophadhyay et al. (2005) investigated the influence of indole-3-acetic acid
(IAA), gibberellic acid and kinetin on growth of Pleurotus sajor-caju (Fr.) Singer in whey. The hormones, at different concentration increased the biomass production of P. sajor-caju by 15 – 26%. Therefore, it might be of interest to investigate if they have an influence on the growth of other mushrooms species also.
Plant hormones are not nutrient, but chemicals that in small amounts are involved in promoting and influence the growth, development, and differentiation of cells and tissues (Öpik and Rolfe, 2005). The substances regulating growth and development of plants consists of five major classes, which are, auxins, cytokinins, gibberellins, abscisic acid (ABA) and ethylene. Auxins, cytokinins and gibberellins are encouraging substances, while ABA and ethylene are hindering ones (Westwood, 1993;
Eri, 1998; Frat, 1998).
Auxins are compounds that are highly effective in the enlargement of cell, bud formation and root initiation. It also initiates the production of other hormones and in conjunction with cytokinins. Auxins, which are formed in meristematic tissues, control the growth of stems, roots, and fruits, and convert stems into flowers (Osborne and
McManus, 2005). Auxins accelerate the elongation and increase the growth and the division of cells. Auxins are light sensitive compounds, which decrease in light and increase in dark. High concentration of auxins in plants will cause the domination of growth at the peaks of the plant, which formed the apical dormancy. Thus, the germination of the side buds is under pressure (Korkutal et al., 2008). IAA is the main naturally occurring auxins in plants, which derived from indole containing a carboxymethyl group (acetic acid) (Zhao, 2010). Plants mainly produce IAA from 7 tryptophan through indole-3-pyruvic acid (Mashiguchi et al., 2011; Won et al., 2011).
IAA is also produced from tryptophan through indole-3-acetaldoxime in Arabidopsis sp.
(Sugawara et al., 2009). IAA, naphthalene acetic acid (NAA) (Dey et al., 2007;
Maniruzzaman, 2004; Alexander and Lippert 1989) and 2,4-dichlorophenoxyacetic acid
(Jonanthan and Fasidi, 2001) are shown to enhance the mycelia growth of mushrooms.
Cytokinins are effective compounds in the regulation of the growth of plants by increasing the division of cells (Korkutal et al., 2008). They were called kinins when the first cytokinins were isolated from yeast cells. The most common types of cytokinins are zeatin, 2ip, benzil adenine (BA) and tetrahydro piranil benziladenine (PBA).
Cytokinins accelerate the division of cells, regulate nucleic acids, they encourage the dominance and branching on the peaks, stimulate the start of bud burst, prevent flowers, fruit and tree from aging and also falling down (Westwood, 1993; Secer,
1989;Güleryüz, 1982). Cytokinins counter the apical dominance induced by auxins; they in conjunction with ethylene promote abscission of leaves, flower parts and fruits
(Deborah and Einset, 1983). 6-benzylaminopurine (BAP), or known as benzyl adenine, is a first-generation synthetic cytokinin that promotes plant growth and development responses, setting blossoms and stimulating fruit richness by stimulating cell division. It is an inhibitor of respiratory kinase in plants, and increases post-harvest life of green vegetables. Kinetin (KIN), a synthetic cytokinin, was reported to increase the biomass and protein content of Agaricus campestris sensu Cooke (Guha and Banerjee, 1974), and yeast Kluyveromyces fragilis (A. Jörg.) Van der Walt (Paul et al., 2002).
Gibberellins include a large range of chemicals that are produced naturally within plants and by fungi. They were first discovered in Japan by a fungus called
Gibberella fujikuroi (Sawada) Wollenw. that produced a chemical, which cause abnormal growth in rice plants (Grennan, 2006). Gibberellins provide elongation of plants by increasing the growth and division of the cells like auxins. The plants, which 8 are rich in gibberellins, have long intermodes. They are less sensitive to light when compared to auxins and they show less depressive effect in high-dose applications.
They encourage germination by breaking the dormancy of the seeds. The completion of the dormancy within the botanical organs is proportional to the amount of increase in gibberellin. Gibberellins are known to increase the parthenocarpic fruit production like auxins and even they are sometimes more efficient (Eri, 1998; Westwood, 1993; Secer,
1989). Paul et al. (2002) reported that gibberellic acid (GA3) increased biomass production of food yeast K. fragilis in deprotenized whey. Gibberellic acid also showed an enhancement of growth and protein content of P. sajor-caju (Mukhopadhyay et al.,
2005; 1999).
Michniewicz (1987) reported that ABA had been found to be strong stimulator of growth and development of Fusarium culmorum (W. G. Sm.) Sacc.. ABA, also known as dormins, has a great role in preventing growth and biophysiological cases.
The most typical effects of these substances are that they prevent germination and bud burst by affecting the division of cells. It is seen in many research that ABA, which is given from outside, induced the closing of the stomata and thus, it hinders transpiration
(Eri, 1998; Secer, 1989; Cimen, 1988).
Ethylene (C2H4) is a simple compound and known to be highly efficient substance in a gas form, which is generated by the plant itself, ethylene could control growth and development and it is produced in all tissues. The principal effects of ethylene on plants are increasing the maturity of the fruit, accelerating the fall of leaf and fruit, regulating bloom, limiting elongation of plants, encouraging rooting on canes and preventing axillary bud formation of the plant (Eri, 1998; Frat, 1998; Westwood,
1993).
9
2.1.2 Fruiting substrate formulations
The physiological condition and nutritional state of the mycelium influence the basidiocarp formation. (Flegg and Wood, 1985; Madelin, 1956). Fermentation process involves cultivation on specific substrates by imitating the natural way of mushroom life
(Tengerdy and Szakacs, 2003). The medium substrates have to be considered based on the species of mushroom. For example, the litter decomposer Agaricus bisporus (J.E.
Lange) Imbach. has been commercially grown on straw supplemented with nitrogen
(manure) composted in two phases (outdoor fermentation and indoor pasteurization) over three weeks (Moore and Chiu, 2001; Fermor et al., 1985; Stamets and Chilton,
1983). On the other hand, the white rot mushroom fungi, such as Pleurotus spp., L. edodus, and F. velutipes, are cultivated on non-composted lignocellulosic substrates, by exploting their ability to produce enzymes to degrade all wood components (Zadrazil et al., 2004; Chen et al., 2000).
Unlike the autotrophic higher plants, which obtain water and inorganic nutrient from soil and synthesize organic compounds in leaves through photosynthesis, mushrooms are heterotrophic organism, which obtains all the required nutrition from the substrate. The mushroom substrate, which is the main source of nutrients, is one of the crucial factors that greatly affect growth and fructification. Most saprotrophic basidiomycetes have relatively simple nutritional requirements for growth, fruiting and ability to withstand microbial competitors (Scrase and Elliott, 1998). However, different species of cultivated mushrooms have different substrate requirements. Madelin (1956) stated that it is important to keep a balance between carbon and nitrogen sources for the induction of fruiting body. The effect of nitrogen is less specific than that of carbon.
The carbon-to-nitrogen ratio (C:N ratio) obtained from chemical analysis of fungal cells is approximately 10:1, but substrate carbon is also used for energy and is respired as carbon dioxide. Thus, Chang and Miles (2004) estimated that an amount is converted to
10 cellular material that is similar to the amount respired as carbon dioxide. Consequently, for growth, a C:N ratio of 20:1 is suitable. Charlesworth (1995) estimated that C:N ratio between 80:1 and 10:1 is suitable for substrate media. Since 1928, the mixture of sawdust (80%) and rice bran (20%) is used commercially as fruiting substrate for F. velutipes (Nakamura, 1981).
Lignocellulosic residues mainly consist of insoluble polysaccharides such as cellulose, lignin, and hemicellulose. Carbon sources provide the structural and energy requirement of the fungal cell (Chang and Miles, 2004). Agroresidues such as cereals straw, cotton stalks, sugarcane baggase, coffee pulp and coffee husk, rice husks, waste paper, wood sawdust and chips, are some examples of carbon sources that can be used as fruiting substrate (Philippoussis, 2009). During vegetative (mycelial) growth, the fungi produce a wide range of extracellular enzymes to degrade the lignocellulosic substrates: peroxidases and laccases for lignin degradation, and glucanases, cellulases and xylanases for cellulose and hemicelluloses degradation (Stoop and Mooibroek
1999; De Groot et al. 1998). There are considerable changes in enzyme activities that occurred during fruiting that indicates a connection to the regulation of basidiocarp development. A bisporus and L. edodes shows laccase activities are highest before the initiation of basidiocarp, and decline rapidly with aggregate formation, whereas cellulase activities are highest during the development of basidiocarp (Ohga et al., 1999;
De Groot et al., 1998; Ohga, 1992).
Nitrogen supplementation may influence crop yield and basidiocarp size.
Nitrogen is essential for the synthesis of proteins, purines, and pyrimidines. Chitin, a polysaccharide of common occurrence in the cell walls of many fungi, also contains nitrogen (Chang and Miles, 2004). Substrates for mushroom cultivation normally contain organic nitrogen sources and low in free ammonium, since excess can inhibit growth or fruiting ability (De Groot et al., 1998; Moore 1998). Wang (2000) recorded 11 that F. velutipes grew well in medium with soy bean powder, peptone, beef cream, and yeast powder as nitrogen sources, but could not grow well with nitrate or amine nitrogen as the nitrogen source.
Kinugawa (1972) stated that inorganic nutrient such as magnesium and phosphorus are effective for the growth of mycelial and the initiation of basidiocarp formation. The phosphate ion is indispensable for fruiting. In addition, the effect of trace elements and vitamin such as thiamine (vitamin B1) are also recognized for mycelial growth and basidiocarp formation.
Production of edible or medicinal mushroom is a successful example of agro- wastes to be recycled (Chiu et al., 2000). Different kinds of agricultural by-products have been used or tried for growing various edible mushrooms in the world. In Nigeria,
Akinyele and Akinyosoye (2005) reported that Volvariella volvacea (Bull.) Singer was able to be cultivated on rice husk, paddy straw, cotton waste, groundnut shell, cassava peel, corncob, white afra dust, red afra dust and oil palm pericarp. Peng (1989) stated that the most extensively used agrowaste for cultivation of edible mushrooms in Taiwan are rice straw, rice bran, wheat bran, cotton waste, chicken manure, and sawdust or wood chip. In India, Ragunathan et al. (1996) reported that P. sajor-caju, P. platypus Sacc. and P. citrinopileatus Singer, were cultivated on various agroresidues such as paddy straw, maize stover, sugarcane bagasse and coir pith.
As the nutrient composition of the substrate is one of the factors limiting colonization as well as quantitative and qualitative yield of cultivated mushroom
(Philippoussis et al., 2002; 2000), supplements containing sugars and starch (easily available carbohydrates) and fats (slower degraded and time-lasting nutrient sources) are added to the basal ingredient. Supplements are used to increase nutritional content, speed-up growth and increase mushroom yield, especially in the cultivation of white rot
12 mushroom (Naraian et al., 2008; Royse 1996; Royse et al., 1990). The various organic supplements used in mushroom cultivation comprises molasses, brewer’s grain, grasses and waste paper, cotton and coffee waste etc (Przybylowicz and Donoghue, 1990).
2.1.3 Mushroom growth
In mushroom growth, there are two phases in the life cycle i.e. the mycelium
(vegetative phase) and the basidiocarp (fruiting body) formation (reproductive phase).
After the inoculation step, the mycelium grows through the substrate by degrading its ingredients and supports the formation of basidiocarps. Mycelial growth and fruiting during this stage are regulated by temperature, gaseous environment, nutrient status, humidity, and in certain cases by light (Zadrazil et al., 2004; Wood 1989). Basidiomata production on the culture medium surface occurs as a series of cycles (flushes).
Biological efficiency (BE) expresses the bioconversion of the dry substrate to fresh basidiocarps and indicates the fructification ability of the fungus utilizing the substrates
(Fan et al., 2000). BE is calculated as the percentage ratio of the fresh weight of harvested mushrooms over the weight of dry substrate at inoculation (Diamantopoulou et al., 2006; Philippoussis et al., 2001; Chang and Chiu, 1992). According to Stamets
(2000), although 250% BE is exceptional, a good grower should operate within 75 –
125% range. After harvesting, mushrooms are normally cooled down to retard basidiocarp metabolism, packed and sent to the fresh market, or processed through freezing, canning or drying depending on marketing strategies (Martínez-Carrera et al.,
2000).
2.2 Flammulina velutipes (Curtis) Singer
Flammulina velutipes is a member of the family Tricholomataceae. It bears the common names as enokitake (Japanese for “The Snow Peak Mushroom”) and
‘cendawan jarum emas’ or ‘golden needle mushroom’ (Malaysia). It is commonly
13 cultivated in regions of temperate climate, which required low-temperature for fruiting
(Lou et al., 1983).
2.2.1 Morphology
Flammulina velutipes is a white rot type of basidiomycete. There is a significant difference in appearance between the wild (Figure 2.1) and cultivated mushroom
(Figure 2.2). Cultivated mushrooms that are not exposed to the light resulted in white colour, whereas wild mushrooms usually have dark brown colour (Sharma et al., 2009).
Colour development is due to the accumulation of phenolic pigments in fruit bodies.
When the mushroom is cultivated in the light, the activity of phenol oxidase increases
(Nakayama et al., 1987). In 1985, a novel strain, M-50, was successfully produced in
Japan. It is the first breed of white basidiocarp forming strains of F. velutipes
(Nakayama et al., 1987; Kitamoto, 1990).
The cultivated mushrooms are also grown to produce long thin stems, whereas wild mushrooms produce a much shorter and thicker stem. The pileus is 2 - 10 cm on wild mushrooms, but under cultivation techniques it is small and more commonly 2 - 3 cm. Initially the pileus, or the cap, is hemispherical in shape, but at maturity it opens to a plane. The surface of the cap is orange-red, yellow tinged at the edges and darker in the center. The gills are white or pale yellow and slightly adnexed (Chang and Miles,
2004).
The stipe is 5 - 10 x 0.4 - 0.8 cm in nature and 2 - 0.9 x 0.2 - 0.8 cm in cultivation and is slightly tapered toward the base. The basidiospores are white, elliptical, smooth and 7 - 10 x 3 - 5 µm in size. The range of sizes and colors varies in the fruiting bodies depending on the conditions in nature, and there are differences between fruiting bodies produced in nature and by the conditions of artificial cultivation
(Chang and Miles, 2004).
14
The hyphae of the monosporous mycelium of F. velutipes have septa and are about 2.1 - 3.2 µm in diameter. The hyphae of the dikaryotic mycelium have clamp connections with branches commonly forming just below the clamps and less commonly above the clamp (Ingold, 1980). Both monokaryotic and dikaryotic hyphae of F. velutipes produce uninucleate oidia (Brodie, 1936).
Figure 2.1 Wild F. velutipes (Source: http://botanofilia.blogspot.com/2011/08/flammulina-velutipes.html)
Figure 2.2 Cultivated F. velutipes (Source: http://en.wikipedia.org/wiki/Enokitake)
15
2.2.2 Nutritional value and medicinal properties of F. velutipes
The efficiency of fungi in converting substrate to protein is far superior to that of several plants and even animals. In general, mushrooms are low in calories, sodium, fat and cholesterol, while rich in protein, carbohydrate, fiber, vitamins and minerals. These nutritional properties make mushrooms a very good dietary food (Buswell and Chang,
1993; Rajaratnam et al., 1993).
Flammulina velutipes is commonly served in soups, stir-fries, salads and other dishes. It have flavorful taste and slightly crisp. Flammulina velutipes contains on fresh weight basis, 89.2% of moisture, 17.6% of crude protein, 1.9% of crude fat, 73.1% of carbohydrate, 3.7% of crude fiber, and 7.4% of ash on the basis dry material (Crisan and
Sands, 1978).
Flammulina velutipes, a delicious mushroom rich in peroxidase, superoxide dismutase and other compounds that can prevent some severe disease like cancer and coronary heart disease. It also contains compounds that prevent as well as cure liver disease and gastroenteric ulcers provided it is taken on a regular basis (Ying, 1987;
Yoshioka et al., 1973). In addition, like many other mushrooms, F. velutipes contains immunodomodulatory (Ko et al., 1995), antitumor (Ikekawa et al., 1982), and cholesterol-lowering substances (Fukushima et al., 2001). Both mycelium and basidiocarp of F. velutipes could be recommended for formulating antioxidative dietary supplements (Bao et al., 2009; 2008).
2.2.3 Environmental factors affecting fruiting of F. velutipes
2.2.3.1 Temperature
Temperature is one of the important factors in the control of mycelial growth and fruit body formation. The temperature extremes (maximum and minimum) are of great importance in determining the survival and distribution of a fungal species in
16 nature (Chang and Miles, 2004). Generally, the mycelium grows in the ranges of 3 –
4⁰C to 33 – 34⁰C. The optimum temperature is between 22 and 26⁰C. The mycelium grows slowly, but does not die when exposed to a temperature of 3 - 4⁰C. On the other hand, at around 34⁰C growth ceases and at over 34⁰C the mycelium is killed instantly
(Tonomura, 1978).
The temperature necessary for primordium formation is between 10 and 20⁰C.
According to Kinugawa and Furukawa (1965), a temperature of 15⁰C is more effective than 5 - 10⁰C for primordium formation. Specifically, at a temperature of 15⁰C, it takes about 15 h for primordium formation, but at 5 or 10⁰C it takes about 48 h. Many study suggested that the optimum temperature for F. velutipes to be fruiting is from 10-15⁰C
(Aschan-Åberg, 1958; Wakita, 1958; Kinugawa and Furukawa, 1965). Gruen (1969) also reported that fruit body growth was better at 16⁰C than at 21⁰C.
2.2.3.2 Moisture and humidity
Moisture of substrates and the humidity of the environmental air also affect the fungal growth. High humidity (90 – 95%) is favourable for pinning and fruiting (Kües,
2000; Kinugawa, 1993; Flegg and Wood, 1985), but the moisture content of the substrate might be even more critical towards contamination. The optimal water content for wooden substrates is 35 – 60% and, for other substrates is 60 – 80%. The lower values reflect the oxygen demand of the fungi in the substratum, balanced against their requirement for water (Ohga, 1999; Scrase and Elliot, 1998; Flegg and Wood, 1985).
2.2.3.3 Oxygen supply
Flammulina velutipes is an aerobic species and, must be supplied with sufficient oxygen. Plunkett (1956) showed that under conditions of continuous exposure of carbon dioxide in the air: (1) pileus diameter decreased with increasing concentration of carbon dioxide (0.06 - 4.90% carbon dioxide); (2) stipe elongation was less sensitive to carbon 17 dioxide than pileus expansion; and (3) stipe elongation and pileus expansion were both prevented by high concentration of carbon dioxide. According to Long (1966), the carbon dioxide inhibition of pileus growth can be limited to the expansion phase of pileus development and not to pileus formation and early growth.
2.2.3.4 Light intensity
Besides temperature, light is believed to stimulate the morphological changes that take place during basidiocarp formation of many basidiomycetes mushroom
(Sakamoto et al., 2002). It has been reported that F. velutipes can form basidiocarps in total darkness (Kinugawa, 1977; Plunkett, 1956, 1953; Aschan, 1954), although these basidiocarps lack mature pilei. It was shown that the diameter of the pileus increases in proportion to the light intensity (up to 100 lx) (Inatomi et al., 2001), and thus it is believed that the formation of the pileus of F. velutipes is stimulated by light. Fruiting in the darkness has also been reported for several other basidiomycetes mushrooms. For example, when Coprinus cinereus (Schaeff.) Gray is grown in complete darkness; it forms basidiocarp with long stipe and a very thiny, undeveloped pileus on top that is denoted as a dark stipe (Tusué, 1969).
2.2.3.5 Hydrogen ion concentration (pH)
pH has great effects on morphological development. The pH requirements for growth and fruiting differ as to their optimal values but not necessarily in the same direction from one species to another (Miles, 1999). Mycelial growth is less affected by pH, but basidiocarp development of several species occurs best at neutral or slightly acidic pH values around pH 6 - 7 (Kinugawa, 1993; Flegg and Wood, 1985). The pH change that occurs during growth (e.g., by the production of organic acids) may trigger the response from vegetative growth to fruiting (Miles, 1999).
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2.2.3.6 Mechanical injury
The onset of basidiocarp development correlates with nutritional exhaustion of the growth substrates. Basidiocarp development for commercial mushroom production is thus often induced by covering compost colonized by vegetative mycelium with a layer of moist peat and chalk, which have only limited nutrient (Scrase and Elliot,
1998). Typically, mycelia of basidiomycetes are not uniformly competent to differentiate; and only young hyphae can be induced to initiate fruiting body development (Ross, 1982). Mechanical injury of established mycelium locally stimulates basidiocarp development, because wounding causes outgrowth of fresh hyphae (Scrase and Elliot, 1998; Granado et al., 1997; Leonard and Dick, 1979). The molecular principles triggering differentiation are not known. Various substances with fruiting inducing activity in specific or several basidiomycetes have been described: cerebrosides (Kües, 2000; Wessels, 1993), sucrose esters of fatty acids and other surfactants (Magae, 1999; Magae and Itoh, 1998; Oita and Yanagi, 1993), cAMP and
AMP (Kües, 2000; Wessels 1993), anthranilic acid and indole (Samadder et al., 1997;
Wessels, 1993) and other substances of yet unknown nature present in fungal extracts
(Butler and Pearce, 1999).
2.3 Agricultural Lignocellulosic Wastes in Malaysia
Mushroom species have the ability to degrade lignocellulosic residues either in original or composted form (Rajarathnam et al., 1998), but they vary in the production of extracellular degradation enzymes and thus, different ability to grow and fruit on agroresidues (Baldrian and Valášková, 2008; Chen et al., 2003; Bushwell et al., 1996).
Kuhad et al. (1997) stated that a huge amount of livestock waste, agricultural crop residues and agroindustrial by-products are annually generated, the major part being lignocellulosic biomass.
19
Wood and wood residues, obtained from the forest and sawmill, are the most popular lignocellulosic agroresidue used as main substrate for commercial production of mushrooms worldwide. Wood residues, such as bark, chips, sawdust, coarse residues, and planer shavings, obtained either from the primary processing and secondary manufacturers. According to Alderman (1998), during the sawing of a log at a typical sawmill, approximately 50% of the initial log volume is converted into wood products and 50% is converted into wood residues. The chemical properties of wood residues vary with the type of wood. Wood chips or sawdust derived from softwood contains
37.7 – 49.5% of cellulose, 10.7 – 25.0% of hemicelluloses, 26.1 – 29.5% of lignin, 0.4 –
0.5% of ash and 0.1% of nitrogen (Tisdale et al., 2006; Palonen, 2004; Ward et al.,
2000). Wood chips or sawdust derived from hardwood contains 42.9 – 45.1% of cellulose, 22.0 – 33.0% of hemicellulose, 24.0 – 26.0% of lignin, 0.2 – 0.3% of ash and
0.1 – 0.2% of nitrogen (Tisdale et al., 2006; Gabriel, 2004; Philippoussis et al., 2001).
In Malaysia, the overall production index of the wood and wood-based products industry increased by 3.6% to 112.8% in 2005 while from 108.9% in 2004. This is mainly due to strong external demand for laminar board, particle board and other panels and boards (Ministry of International Trade and Industry, 2006). However, the supply of these resources is declining. The export of rubber wood swan timber is banned to ensure adequate supply of rubberwood for wood industry. These caused the limited supply of wood residues, especially sawdust, and furthermore, the competition in usage of sawdust between mushroom cultivation with other industries is increasing.
Except cedars and redwoods, not all wood are recommended to be used as the source of mushroom growing substrate as they decompose slowly due to their anti- rotting compounds, and hence stifle mushroom growth (Stamets, 2000). Most of the wood industries run mixed wood and do not separate their sawdust into identifiable piles, which give an obstacle for mushroom growers. Beside that sawdust supplies are 20 often mixed up with chemicals either during processing or transportation due to the heavy metal contamination (Stamets, 2000) that can affect mushroom growth.
There are many lignocellulosic agroresidues produced in Malaysia such as paddy straw, rice husk, sugarcane baggasse, oil palm frond, sago ‘hampas’ and cotton stalk that are potential substrates for mushroom cultivation (Sabaratnam et al., 2006).
Agroresidues derived from rice cultivation and palm oil processing industries are potential substrates to be used as an alternative substrate. Spent yeast derived from brewery industry contains high nitrogen content which is suitable as the supplement, similar to rice bran.
2.3.1 Agroresidues derived from rice cultivation
Rice (Oryza sativa L.), which belongs to the family Graminae, is the most important cultivated cereal crop worldwide. It is the world’s most important food crop and primary food source for more than a third of world’s population (Kamal et al.,
2009). In Malaysia, rice production of 1 619.2 MT, is in the second ranked after palm oil, 17 564.9 MT (Department of Statistics, 2011). Rice is the staple food in Malaysia.
In the year of 2010, it was reported that 2, 464, 831 MT of paddy and 1, 588, 457 MT of rice were produced (Department of Statistic, 2011).
More than one million tonnes of paddy straw are produced annually in
Peninsular Malaysia (Puad et al., 2010). Paddy straw, rice husk and rice bran are agroresidues derived from rice cultivation. Paddy straw is the dry stalks of rice plants, after the grain and chaff have been removed. Due to its abundance, the local farmers usually burned the paddy straw once dried in the field, and only a small portion of the residue is reserved as animal feed. This caused haze and other environmental problems
(Liu et al., 2009). There are biotechnology approaches to utilize paddy straw as biofuel
(Binod et al., 2010), mulching mat for weed control (Alloub, 2001), fibre board (Yang
21 et al., 2003), and in paper-making (Hoang et al., 2001). Compared to empty fruit bunches (EFB) and palm pressed fiber (PPF), paddy straw had been used as mushroom fruiting substrate. Madan et al. (1987) and Bisaria et al. (1987) reported that one dry ton of paddy straw would yield about 1000kg of the oyster mushroom. Ho and Peng (2006) also reported that paddy straw has been used as the main carbon source substrate for A. bisporus, and A. bitorquis (Quél.) Sacc. in Taiwan. However, there are lacks of awareness among the Malaysian mushroom growers in using paddy straw as fruiting substrate.
Paddy straw is a good source of carbon, while rice bran contains high nitrogen.
Wati et al. (2007) stated that paddy straw is produced abundantly as a lignocellulosic by-product of rice crop with an annual worldwide production of 800 MT. More than 1 million MT of paddy straw are produced annually in Peninsular Malaysia (Puad et al.,
2010). Paddy straw is the dry stalks of rice plants, after the grain and chaff have been removed. Paddy straw contains bound sugars such as cellulose (22.8 – 38.4%) and hemicelluloses (17.7 – 28.5%) meshed with lignin (6.4 – 18.0%), with ash content of
8.3 – 17.8% (Paranthaman et al., 2010; Mata and Savoie, 2005; Howard et al., 2003).
The utilization of paddy straw as an alternative material for mushroom growing substrate is important to solve the environmental pollution problems associated with open-field burning and soil incorporation.
However, the nutrient composition of the substrate is limiting to the yield of cultivated mushroom (Philippoussis et al., 2002; 2000), supplement with protein-rich
(nitrogenous) material are needed to enhance the base substrate (Stamets, 2000). Rice bran is the most popular and available organic supplement for growing a number of edible mushrooms in Asia (Peng et al., 2000). Rice bran, a hard outer layer of grain that consists of combined aleurone and pericarp, is a by-product material derived from the
22 rice milling process. It contains 37% of carbohydrates, and 2.0% of nitrogen
(Przybylowicz and Donoghue, 1990).
2.3.2 Agroresidues derived from palm oil mill
Oil palm (Elaeis guineensis Jacq.) production dominated the crops sub-sector with a share of 76.9% in 2008, which indicates that oil palm is the main commodity in
Malaysia (Ministry of International Trade and Industry, 2006). There are more than three million hectares of oil palm plantations (Lim, 2000). Approximately 90 million
MT in total of renewable biomass derived from palm oil industry are produced each year. Prasertsan and Prasertsan (1996) stated that there are various forms of solid and liquid wastes from the mill, which include EFB, PPF, palm kernel cake (PKC), palm kernel shell (PKS), sludge cake (SC) and palm oil mill effluent (POME). In this study,
EFB and PPF are chosen as the subjects to investigate the suitability for mushroom growth.
Palm oil is one of the major primary commodities in Malaysian economy with
18, 300, 000 MT production (Ministry of Finance, 2012a). Malaysian Palm Oil Board
(MPOB) (2012) stated that the yield of fresh fruit bunches (FFB) for January – June
2012 was 7.84 MT/hectare. Chan (1999) reported that every tone of FFB processed, 220 kg of empty fruit bunches (EFB) which is the major component of all solid waste of palm oil mill. He also reported that from 1 ha of land, about 1.63 MT of dry palm pressed fiber (PPF) are generated. Most of these lignocellulosic agroresidues were disposed of through incineration and dumping. With only a small portion used by the local mills for their heat and power requirement, and for mulching and soil conditioning or fertilizer.
EFB is the major component of all solid waste, which consists 20 – 30% of the fresh fruit bunches (FFB) composition (Prasertsan and Prasertsan, 1996). EFB is the
23 residue left after the FFB are pressed at oil mill to extract the oil. Due to its high moisture content of 60% that resulted from steam during sterilization, makes it unsuitable as fuel. It was reported that the EFB has 42% carbon, 0.8% nitrogen, 0.06% phosphorus, 2.4% potassium and 0.2% magnesium (Krause, 1994). This fibrous material shows good potential to be used as mushroom growing substrate, without any treatment, such as V. volvacea (Prasertsan and Prasertsan, 1996) and P. sajor-caju
(Muhamad et al., 2008).
Unlike EFB, the oil retained in its cell wall makes the PPF a good combustible material. The PPF contains 1.7 – 6.6% phosphorus, 17 – 25% potassium and 7% calcium (Krause, 1994). Similar to paddy straw, PPF contains a higher percentage of fiber and lignin, which makes it a good substrate for mushroom cultivation such as P. sajor-caju (Klitsaneepaiboon and Bunkong, 1990).
2.3.3 Brewery solid waste
In brewing, surplus yeast is recovered by natural sedimentation at the end of the fermentation and conditioning. Only part of the yeast can be reused as new production yeast. Spent yeast is very high in protein and vitamin B (Goldammer, 2008), which is a great potential candidate to be used as nitrogen supplement in mushroom cultivation.
24
CHAPTER 3.0 MATERIALS AND METHODS
3.1 Flammulina velutipes Culture
Flammulina velutipes (KUM60375) culture used in this study was maintained on malt extract agar (MEA) (as described in Appendix A). Stock culture was maintained on
MEA slants and sterile water deposited at Mushroom Research Centre culture collection, Faculty of Science, University of Malaya, Kuala Lumpur.
3.2 Effect of Plant Growth Hormones on Mycelial Growth of F. velutipes on MEA
3.2.1 Preparation of mycelium culture and measurement of growth
Flammulina velutipes culture grown on MEA in Petri dishes for 7 days was cut at the periphery of the colony using sterile 7 mm diameter cork borer and used as inoculum. One mycelium plug was centrally inoculated onto each solidified MEA in
Petri dishes supplemented with plant growth hormones at concentrations as described in
3.2.2. The plates were then incubated at 25˚C. The hormones solution was prepared by dissolving in 0.1 M NaOH. The plant growth hormones used in this study were 6- benzylaminopurine (BAP) and β-indole acetic acid (IAA) which obtained from Sigma
Chemical Co.. Mycelium growth in each Petri dish was determined by measuring the average diameter of the mycelium colony every day for 10 days. The average reading was plotted against time (day) to obtain the growth rate in mm/day. Analysis of the design of experiment is done based on the analysis of variance (ANOVA), a collection of models in which the observed variance is partitioned into components due to the difference factors which are estimated and/or investigated.
3.2.2 Experimental design to determine the effect of hormone on mycelial growth
The effect of plant growth hormones on mycelial growth of F. velutipes was investigated by full factorial design generated using MINITAB® version 14 (2004). To set a mathematical model between responses and factors, one response was under
25 investigation for mycelial growth: mm/day. Two factors (consisting of two plant growth hormones) that showed effective response were chosen in this study, namely: BAP and
IAA. For each of the factor, two different levels were set, which corresponded to low and high levels of treatment conditions. Factors and levels were given in Table 3.1.
Table 3.1 Experimental factors and levels for screening Parameter values: Concentrations (mg/L) Factors Low level High level BAP 1.0 10.0 IAA 1.0 10.0 The experimental data obtained for the response variable studied. Plant growth hormones used as are 6-benzylaminopurine (BAP) and β-indole acetic acid (IAA).
Complete trials represented all possible combinations of the factors to determine the optimum concentration of hormones that affect the mycelia growth. Fifteen treatments (runs) were carried out whereby the growth media were supplemented with various combination concentrations (mg/L) of plant hormones, as shown in Table 3.2.
26
Table 3.2 The experimental design of various combination concentrations of plant growth hormones Standard Center Concentration (mg/L) Run order Blocks order point BAP IAA 2 1 1 1 10.0 1.0 13 2 0 1 5.5 5.5 4 3 1 1 10.0 10.0 3 4 1 1 1.0 10.0 1 5 1 1 1.0 1.0 15 6 0 1 5.5 5.5 11 7 1 1 1.0 10.0 7 8 1 1 1.0 10.0 6 9 1 1 10.0 1.0 8 10 1 1 10.0 10.0 5 11 1 1 1.0 1.0 9 12 1 1 1.0 1.0 12 13 1 1 10.0 10.0 10 14 1 1 10.0 1.0 14 15 0 1 5.5 5.5 The experimental data obtained for the response variable studied. Plant growth hormones used as are 6-benzylaminopurine (BAP) and β-indole acetic acid (IAA).
Based on the above results, the response surface method (RSM) was used in the optimisation of hormone concentrations using the central composite design (CCD). The maximum range was set at 1.5 mg/L and the minimum at 0.5 mg/L of the hormones concentration (as mentioned in Table 3.3). The range was selected whereby the concentration that gave the highest mycelial growth rate above (1.0 mg/L each plant growth hormones) was the centre point. There were 33 experimental runs including triplicates generated using MINITAB® 14 as shown in Table 3.4.
Table 3.3 Experimental factors and levels for optimisation Parameter values: Concentrations (mg/L) Factors Low level High level BAP 0.5 1.5 IAA 0.5 1.5 The experimental data obtained for the response variable studied. Plant growth hormones used as are 6-benzylaminopurine (BAP) and β-indole acetic acid (IAA).
27
Table 3.4 The experimental design of various combination concentrations of plant growth hormones for optimisation Standard Center Concentration (mg/L) Run order Blocks order point BAP IAA 28 1 -1 1 1.5 1.0 6 2 -1 1 1.5 1.0 4 3 1 1 1.5 1.5 2 4 1 1 1.5 0.5 30 5 -1 1 1.0 1.5 24 6 1 1 1.5 0.5 13 7 1 1 1.5 0.5 11 8 0 1 1.0 1.0 14 9 1 1 0.5 1.5 9 10 0 1 1.0 1.0 19 11 -1 1 1.0 1.5 25 12 1 1 0.5 1.5 23 13 1 1 0.5 0.5 29 14 -1 1 1.0 0.5 26 15 1 1 1.5 1.5 8 16 -1 1 1.0 1.5 16 17 -1 1 0.5 1.0 12 18 1 1 0.5 0.5 27 19 -1 1 0.5 1.0 33 20 0 1 1.0 1.0 17 21 -1 1 1.5 1.0 18 22 -1 1 1.0 0.5 3 23 1 1 0.5 1.5 15 24 1 1 1.5 1.5 20 25 0 1 1.0 1.0 10 26 0 1 1.0 1.0 5 27 -1 1 0.5 1.0 32 28 0 1 1.0 1.0 7 29 -1 1 1.0 0.5 31 30 0 1 1.0 1.0 21 31 0 1 1.0 1.0 22 32 0 1 1.0 1.0 1 33 1 1 0.5 0.5 The experimental data obtained for the response variable studied. Plant growth hormones used as are 6-benzylaminopurine (BAP) and β-indole acetic acid (IAA).
The response optimiser was applied by using the MINITAB® 14 for determining the exact optimum level of independent variable leading to individual and overall responses. For validation, experimental data were compared with predicted values in
28 order to verify the adequacy of final reduced models. Close agreement and no significant difference should exist between the experimental and predicted values.
3.3 Selection of Various Lignocellulosic Agroresidues as The Base Carbon-source for Fruiting Substrate of F. velutipes
3.3.1 Preparation of alginate immobilised mycelium of F. velutipes as inoculum
Alginate-immobilised mycelium was used to replace the grain spawn. Mycelium of F. velutipes was grown on MEA supplemented with 0.5 mg/L BAP+0.5 mg/L IAA for one week. For the preparation of alginate-immobilised mycelium, mycelial discs were inoculated together with malt extract (ME) broth containing alginic acid, and then placed in 0.25 M calcium chloride solution to be encapsulated. After 15 min, the immobilised mycelium was rinsed twice with sterile distilled water to remove impurities on the surface of the support material.
3.3.2 Selection of fruiting substrates
The agroresidues studied were sawdust (SD), paddy straw (PS), empty fruit bunches (EFB), and palm press fiber (PPF) (Figure 3.1), and their carbon and nitrogen content composition is shown in Table 3.5. The C:N ratio of the agroresidues is often used as a relative reference to characterize compost. SD was obtained from Bangi, situated on the south of Hulu Langat, Selangor. PS was collected from Sungai Besar, in the district of Sabak Bernam, Selangor. EFB and PPF were acquired from Seri Ulu
Langat Palm Oil Mill Sdn. Bhd., in Dengkil, Selangor.
29
Table 3.5 The percentage of carbon and nitrogen in lignocellulosic by-products used as fruiting substrates Sample Carbon (C) (%) Nitrogen (N) (%) C:N Sawdust (SD) 85.25 0.90 94.72 Paddy straw (PS) 77.40 0.70 110.57 Empty fruit bunches (EFB) 89.71 0.36 249.19 Palm-pressed fibre (PPF) 84.25 0.60 140.42
The percentage of carbon of was tested by using the Furnace method. The Kjeldahl method was used to determine the percentage of nitrogen.
SD PS
EFB PPF
Figure 3.1 Carbon-source substrates: SD (sawdust), PS (paddy straw), EFB (empty fruit bunches), and PPF (palm pressed fiber).
All the agroresidues were dried and ground into fine particles as shown in Figure
3.1. In order to determine suitable substrates and composition ratios for the cultivation of F. velutipes, various agroresidues and combinations were tested. In the initial selection, single and combinations of agroresidues were tested by preparing the substrates in Petri dishes to determine the radial growth rate of mycelial. Based on the result from the first step, the selected formulations of substrates were then tested by
30 preparing the substrates for fruiting body formation in polypropylene plastic bags to determine the total yield of basidiocarps and biological efficiency (BE).
3.3.2.1 Mycelial growth on various substrate formulations in Petri dishes
Each agroresidue (SD, PS, EFB, and PPF) was tested singularly (100) as control.
In addition, there were six combinations of each pairs of agroresidues at a ratio of 3:1
(75:25), 1:1 (50:50) and 1:3 (25:75), as listed below:
i. SD + PS
ii. SD + EFB
iii. SD + PPF
iv. PS + EFB
v. PS + PPF
vi. EFB + PPF
Singular and combination substrate formulations were investigated to determine the effect of agroresidues on mycelial growth and basidiocarp formation. The substrates were thoroughly mixed. The distilled water was added until the substrate was moistened to at least 80%. Calcium carbonate (CaCO3) or acetic acid was added to adjust the pH to
6. The substrate medium (20 g) was then transferred to Petri dishes (100 x 20 mm) and sterilised by autoclaving twice at 121˚C and 15 psi for 1 h. Three replicates were performed for each substrate formulation. After cooling the substrates to room temperature, they were inoculated with a bead of mycelium inoculum. Inoculated Petri plates were incubated at 25 – 28⁰C to be colonised by the mycelium. Mycelial growth was measured as in 3.2.1. Results were evaluated by one-way analysis of variance
(ANOVA) using Minitab® 14 statistical software.
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3.3.2.2 Mycelial growth and basidiocarp yield on selected substrate formulations in bags
Six formulations were selected viz PPF (100), SD+PS (50:50), SD+PPF (75:25),
EFB+PPF (25:75), PS+EFB (25:75), PS+PPF (50:50) and SD+EFB (50:50), to determine the mycelial growth and basidiocarp yield in polypropylene plastic bags (82 x
322 mm). The preparation of substrate medium was as described in 3.3.1. Bags were filled with the substrates to a height of 10 cm and the weight of the bags was recorded.
Three replicates were prepared for each substrate formulation. The bags were then capped and sterilised twice in an autoclave at 121˚C and 15 psi for 1 h. After sterilised bags were cooled to room temperature, they were inoculated with three mycelium beads. The inoculation was done under aseptic condition. Then, the inoculated bags were incubated at an ambient temperature of 25˚C (±2˚C) until full spawn run completed. The length of the mycelium run was measured daily in unit of millimeter
(mm). For induction of fruiting, the cap was removed and the top of the bag was folded down and the surface of substrate medium was raked to a depth of 2 cm. Subsequently, the bags were placed in the incubator at 8˚C with a humidity of 60 - 70% to stimulate primordia formation. Once primordia formed, the temperature was increased to 15˚C until the stipe was approximately 2 cm in length. When the stipes had elongated within
2 to 3 cm from the substrate, a paper was placed around the bag to form almost cylindrical shaped. The paper was removed when the basidiocarps had matured enough
(at least the stipes is around 13 to 14 cm long). The basidiocarps were harvested and, the yield of mushroom were recorded every flushes. Mycelium thickness was also observed. The biological efficiency (BE) was determined as the following formula:
( ) Biological efficiency, BE (%) = ( ) Equation 3.2 Biological efficiency
32
3.4 Effect of Different Levels of Nitrogen-source Supplementation on Selected
Substrate Formulations on Yield of F. velutipes.
3.4.1 Preparation of fruiting substrates supplemented with nitrogen-source
Rice bran (RB) and spent yeast (SY) were used as the nitrogen-source supplement (Figure 3.2). Rice bran was obtained from Bangi, situated on the south of
Hulu Langat, Selangor while spent yeast was obtained from Carlsberg Brewery
Malaysia Bhd., in Shah Alam, Selangor. The nitrogen content of rice bran and spent yeast are given in Table 3.6.
Table 3.6 The percentage of carbon and nitrogen in rice bran (RB) and spent yeast (SY) Sample Carbon (C) (%) Nitrogen (N) (%) Rice bran (RB) 80.92 2.00 Spent yeast (SY) 25.63 2.00
The percentage of carbon in the samples was analysed using the Furnace method. The Kjeldahl method was used for the percentage determination of nitrogen.
RB SY
Figure 3.2Nitrogen-source supplements used: rice bran (RB) and spent yeast (SY)
To determine the optimum concentration of nitrogen-source supplementation, three combinations of carbon-source substrates were chosen to be the main substrate medium for this study: PS+EFB (25:75), PS+PPF (50:50), and PPF (100). As for the control of this study, the commercialized combination of SD+RB (80:20) was used. The preparation of fruiting substrate was the same as in the previous study (3.3.2.2). The concentration of the nitrogen-source was measured as a percentage of the total weight of
33 the agroresidues substrate. The substrate pH was fixed to pH 6. The length of the mycelium run was measured daily in unit of millimeter (mm). The yield of basidiocarps were recorded every flushes, and the biological efficiency (BE) were calculated.
3.4.2 Experimental design for nitrogen supplementation
To design the effect of rice bran and spent yeast as supplementation for fruiting substrate, factorial design was used. To set a mathematical model between response and factors, one response was under investigation for mycelia growth: mm/day. Two factors
(nitrogen-source supplements) that showed effective response were chosen in this study, namely: RB and SY. For each of the factor, two different levels were set, which correspond to low and high levels of treatment conditions. Factors and levels were given in Table 3.7.
Table 3.7 Experimental factors and levels Parameter values: Concentrations (%) Factors Low level High level RB 5.0 20.0 SY 5.0 20.0 The experimental data obtained for the response variable studied.
The length of the mycelium run was measured daily in unit of millimeter (mm).
Results were evaluated by one-way analysis of variance (ANOVA) and Duncan tests to show significance among differences if any at 95% level. A 2 x 2 full factorial design was carried out to establish the mathematical relationships and to represent how the rate of mycelium run depends on the percentage of supplementation of RB and SY. An experimental matrix was shown in Table 3.8. Running order for each run was randomized in order to minimize possible systematic errors.
34
Table 3.8 The experimental design of various combination concentrations of nitrogen- source substrates Factors (%) Standard Center Run order Blocks order point RB SY 8 1 1 1 20.0 20.0 2 2 1 1 20.0 5.0 11 3 1 1 5.0 20.0 9 4 1 1 5.0 5.0 12 5 1 1 20.0 20.0 5 6 1 1 5.0 5.0 10 7 1 1 20.0 5.0 15 8 0 1 12.5 12.5 1 9 1 1 5.0 5.0 3 10 1 1 5.0 20.0 7 11 1 1 5.0 20.0 4 12 1 1 20.0 20.0 6 13 1 1 20.0 5.0 13 14 0 1 12.5 12.5 14 15 0 1 12.5 12.5 The experimental data obtained for the response variable studied. Materials used as are rice bran (RB) and spent yeast (SY).
3.5 Statistical Analysis
One-way ANOVA was used to analyze the data to establish significant difference between the means (p = 0.05). All the calculations were performed using
Minitab® version 14 (2004) statistical software.
35
CHAPTER 4.0 RESULTS
4.1 Effect of Growth Hormones on Mycelial Growth of F. velutipes for The
Preparation of Spawn
In this study, the experiment to investigate the effect of growth hormone of F. velutipes mycelial growth was designed based on full factorial, whereby BAP and IAA were chosen as the factors and 1.0 mg/L as low while 10.0 mg/L as high concentration.
Table 4.1 showed the response in terms of the average growth rate of F. velutipes mycelium grown on MEA supplemented with various combination concentrations
(mg/L) of growth hormones. The highest mycelial growth rate occurred on MEA supplemented with 1.0 mg/L BAP+1.0 mg/L IAA, at 10.50±0.06 mm/day, while the lowest growth rate occurred on MEA supplemented with 10.0 mg/L BAP+1.0 mg/L
IAA, of 10.06±0.01 mm/day. Overall, the supplemented MEA significantly enhanced the growth rate of F. velutipes mycelium compared with the unsupplemented MEA
(p≤0.05).
Table 4.1 Screening: Growth rate of F. velutipes mycelium grown on MEA supplemented with different plant growth hormones concentrations BAP (mg/L) IAA (mg/L) Mean Growth Rate (mm/day) 0.0 0.0 7.83±0.06a 1.0 1.0 10.50±0.06b 5.5 5.5 10.34±0.03c 10.0 10.0 10.19±0.04d 1.0 10.0 10.09±0.07de 10.0 1.0 10.06±0.01e The experimental data obtained for the response variable studied. Growth hormones used are 6-benzylaminopurine (BAP) and β- indole acetic acid (IAA). Each value is expressed as mean±standard deviation of five replicates. The homogeneous group is represented in alphabet; same letters denotes insignificant statistical differences (p≤0.05).
Table 4.2 showed that the main effects for BAP (0.000), IAA (0.001), and the
BAP*IAA interaction (0.000) were highly significant, whereby their p-values were less than 0.05. The relative strength of effect, BAP (-0.1667) and IAA (-0.1367) showed low level of hormone concentration resulted in higher mycelial growth rate than high level of hormone concentration. The BAP*IAA interaction (0.2700) showed vice versa.
36
Based on Table 4.3, the p-value for the set of two-way interaction (0.000) was less than
0.05. Therefore, evidence exists that the effect of one factor depends on the level of another factor and the significant effect. In Table 4.2, the coefficient of determination,
R-square value (R2) was given as 0.944 indicating a high correlation between the experimentally observed and predicted values. The R2 (adjusted) was given as 0.922.
This indicates that the percentage of fit between the experimental results and the model result was exceptional (predicted by MINITAB® 14). The closer the R2 value is to
1.000, the stronger the model is, and the better it predicts the response. This was supported by low value of standard deviation of error abbreviated as “S” (0.049). This showed the model can be used to explain most of the variations in the data. The experimental data were fitted to a second-order polynomial as shown in equation below:
2 2 y = b0 + b1x1 + b2x2 + b11x1 + b22x2 + b12x1x2
Equation 4.1 Second-order polynomial where y is the estimated response, b0 is a constant, bi are coefficients for each term, and xi are factors in coded values.
Table 4.2 Estimated effects of growth hormones, coefficients, t-value and p-value for mycelial growth rate (mm/day) Coefficient's Term Effect Coefficient T P Standard Error Constant 10.2100 0.01400 729.08 0.000
BAP -0.1667 -0.0833 0.01400 -5.95 0.000 IAA -0.1367 -0.0683 0.01400 -4.88 0.001 BAP*IAA 0.2700 0.1350 0.01400 9.64 0.000 Center point 0.1300 0.03131 4.15 0.002
S = 0.04851122 R2 = 94.43% R2(adjusted) = 92.20% Growth hormones used as are 6-benzylaminopurine (BAP) and β-indole acetic acid (IAA)
37
Table 4.3 Analysis of variance (ANOVA) for mycelial growth rate (mm/day) on the supplemented MEA with growth hormones Source DF Seq SS Adj SS Adj MS F P Main Effects 2 0.13937 0.139367 0.069683 29.61 0.000 2-Way Interactions 1 0.21870 0.218700 0.218700 92.93 0.000 Curvature 1 0.04056 0.040560 0.040560 17.24 0.002 Residual Error 10 0.02353 0.023533 0.002353
Pure Error 10 0.02353 0.023533 0.002353
Total 14 0.42216 where DF = degrees of freedom Seq SS = sequential sum of squares Adj SS = adjusted sum of squares Adj MS = adjusted mean squares
Figure 4.1 showed four different graphs for residual analyses. Residual values are derived from experimental values deducted by the model fitted values. The normal probability plot of residuals (Figure 4.1a) was far from the straight line. It seems that the normality assumption was satisfied by this data. From Figure 4.1b suggested that the data are homogenized. Residuals versus the fitted values were used to examine non- constant variance, missing higher-order terms, and outliers. In theory, the residuals should be scattered randomly around zero. As for this case, most of the residuals fall within the range of 0.05 and -0.05, which indicated that there was only two residual data exceeded the range and were considered as outliers. The histogram of the residual
(Figure 4.1c) detected multiple peaks, outliers, and non-normality signifies that the data was highly distributed at 0 (symmetric and bell-shaped). From Figure 4.1d, the data suggested that there were outliers at observation order 7 with residual -0.08 (as mentioned in the statistical analysis, Appendix D). This graph was used to detect time- dependence of residuals. Thus, the graph displays that the order of execution of the experiment had no influence on the responses obtained as indicated by the absence of any systematic pattern in plot of standardized residual data points against observation order.
38
A pareto chart of the effects is another useful tool that can be used to compare the relative magnitude and the statistical significance of both main and interaction effects. Figure 4.2 showed that the supplementation of growth regulators were significant effects (α = 0.05) as the parameters (IAA, BAP, and IAA*BAP) passed the significant level line of 95% at 2.23, as proved in Table 4.2. The interaction of
IAA*BAP showed great effect compared with singular IAA and BAP. A main effect plot is an outcome plot that can show consistent difference between levels of a factor.
From Figure 4.3, both lines of the hormones showed that there were stronger effects on the greater slope of line. This indicated that the response mean i.e. mycelial growth rate was dependent on the factor level i.e. concentration of hormones. At 1.0 mg/L BAP+1.0 mg/L IAA supplementation showed greater mean mycelial growth rate, while 10.0 mg/L BAP+10.0 mg/L IAA showed lower growth rate. Thus, low level concentration
(1.0 mg/L) of BAP and IAA were chosen for the optimization of hormone concentration. Based on the results shown, the ratio 1:1 of IAA and BAP was identified as the optimal ratio selected for further study.
Residual Plots for Results (a) Normal Probability Plot of the Residuals (b) Residuals Versus the Fitted Values 99 0.05 90
l
t
a
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u
e 0.00
d
c 50 i
r
s
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e
P R -0.05 10 1 -0.10 -0.10 -0.05 0.00 0.05 0.10 10.1 10.2 10.3 10.4 10.5 Residual Fitted Value (d) (c) Histogram of the Residuals Residuals Versus the Order of the Data 4 0.05
y 3
l
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a
n
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e 0.00
d
u i
2 s
q
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r R F -0.05 1 0 -0.10 -0.08 -0.04 0.00 0.04 0.08 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Residual Observation Order Figure 4.1 Residual plot for F. velutipes supplemented with IAA and BAP
39
Pareto Chart of the Standardized Effects (response is Results, Alpha = .05) 2.23
F actor Name A BA P AB B IA A
m
r A
e
T
B
0 2 4 6 8 10 Standardized Effect
Figure 4.2 Pareto chart of standardized effects for F. velutipes supplemented with IAA and BAP Growth hormones used are 6-benzylaminopurine (BAP) and β-indole acetic acid (IAA)
Main Effects Plot (data means) for Growth rate (mm/day)
BAP IAA Point Type 10.35 Corner Center
)
y
a
d
/ 10.30
m
m
(
e
t
a
r
h 10.25
t
w
o
r
G
f
o 10.20
n
a
e
M
10.15
1.0 5.5 10.0 1.0 5.5 10.0 Concentration (mg/L) Figure 4.3 Main effects plot (data means) for mycelial growth rate of F. velutipes supplemented with IAA and BAP Growth hormones used are 6-benzylaminopurine (BAP) and β-indole acetic acid (IAA)
4.1.1 Optimization of hormone concentrations
The results above were then calculated through the MINITAB® 14 software to further optimize the concentration of hormone to be used. The response surface method
(RSM) was used in this optimization phase by analyzing the central composite design
40
(CCD). The maximum range was set at 1.5 mg/L and the minimum at 0.5 mg/L of the hormones concentration. The range was defined by setting the concentration that gave the highest mycelial growth rate (1.0 mg/L BAP+1.0 mg/L IAA) as centre point. Table
4.4 showed the highest mycelial growth rate was found on MEA supplemented with 1.0 mg/L BAP+1.0 mg/L IAA which was 10.51±0.09 mm/day. The value was non- significantly different with MEA supplemented with 1.0 mg/L BAP+0.5 mg/L IAA, and
0.5 mg/L BAP+0.5 mg/L IAA (p<0.05). The lowest mycelial growth rate was found on the MEA supplemented with 1.5 mg/L BAP+1.0 mg/L IAA (9.40±0.15 mm/day).
Table 4.4Optimization of growth hormone concentration (mg/L) on the growth rate of F. velutipes mycelium (mm/day) BAP (mg/L) IAA (mg/L) Mean Growth Rate (mm/day) 1.5 1.5 9.84±0.01b 1.5 1.0 9.40±0.15a 1.5 0.5 9.97±0.17b 1.0 1.5 10.09±0.15bc 1.0 1.0 10.51±0.09d 1.0 0.5 10.50±0.05d 0.5 1.5 10.12±0.34bc 0.5 1.0 10.16±0.43bc 0.5 0.5 10.39±0.13cd The experimental data obtained for the response variable studied. Growth hormones used are 6-benzylaminopurine (BAP) and β- indole acetic acid (IAA). Each value is expressed as mean ± standard deviation of five replicates. The homogeneous group is represented in alphabet; same letters denotes insignificant statistical differences (p≤0.05).
Table 4.5 showed that the main effects of BAP (0.000) and IAA (0.025) were highly significant where the p-values were less than 0.05. As for the quadratic effect, only the interaction BAP*BAP (0.000) shows significant effect on mycelial growth rate.
The high values of R2 (65.9%) and R2 adjusted (59.6%), and with low value of error terms, S (0.2418) showed that the model can be used to explain the variation in the data.
A second-order regression model was then used to express the results obtained in Table
4.5. The result of analysis of variance (ANOVA) was summarized in Table 4.6. The
ANOVA for growth rate of F. velutipes mycelium showed significant statistical values for linear and square effects at f-value equal to 11.86 and 14.07, respectively, and the p- 41 value were less than 0.05 for both effects. This indicated that the linear and square terms were important in the reduced regression. Statistical plots for analyses of experimental data were also constructed (Figure 4.4).
Table 4.5 Estimated regression coefficients, t-value and p-value for mycelial growth rate (mm/day) Coefficient's Term Coefficient T P Standard Error Constant 10.4024 0.07161 145.26 0.000
BAP -0.2423 0.05699 -4.251 0.000
IAA -0.1354 0.05699 -2.376 0.025
BAP*BAP -0.4604 0.08771 -5.249 0.000
IAA*IAA 0.0576 0.08771 0.657 0.517
BAP*IAA 0.0367 0.06980 0.525 0.604
S = 0.2418 R2 = 65.9% R2(adjusted) = 59.6% Growth hormones used are 6-benzylaminopurine (BAP) and β-indole acetic acid (IAA)
Table 4.6 Analysis of variance (ANOVA) for mycelial growth rate (mm/day) on the supplemented MEA with growth hormones Source DF Seq SS Adj SS Adj MS F P Regression 5 3.04817 3.04817 0.60963 10.43 0.000 Linear 2 1.38652 1.38652 0.69326 11.86 0.000 Square 2 1.64552 1.64552 0.82276 14.07 0.000 Interaction 1 0.01613 0.01613 0.01613 0.28 0.604 Residual Error 27 1.57848 1.57848 0.05846
Lack-of-Fit 3 0.71485 0.71485 0.23828 6.62 0.002 Pure Error 24 0.86362 0.86362 0.03598
Total 32 4.62665 where DF = degrees of freedom Seq SS = sequential sum of squares Adj SS = adjusted sum of squares Adj MS = adjusted mean squares
From Figure 4.4a, the normal probability plot of residuals showed that the residuals were normally distributed, as the residuals were plotted not far from the straight line. By observing the residuals against fitted values (Figure 4.4b), most of the residuals fall within the range of 0.05 and -0.05. This indicated that there was not much difference between the residual results (experimental) and the model results (predicted).
The histogram of the residuals (Figure 4.4c) signifies that the data was highly distributed at 0.2, which showed negative skewed. It means that the distribution was 42 asymmetrical (unbalanced) and the median was larger than the mean. The residual versus the order of the data (Figure 4.4d) suggested that there was outliers at observation order 19 with residual -0.521 (as mentioned in the statistical analysis,
Appendix D).
Residual Plots for Growth rate (a) Normal Probability Plot of the Residuals (b) Residuals Versus the Fitted Values 99 0.5 90
l
t
a
n 0.0
u
e
d
c 50 i
r
s
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P R -0.5 10 1 -1.0 -0.5 0.0 0.5 9.8 10.0 10.2 10.4 10.6 Residual Fitted Value (c) Histogram of the Residuals (d) Residuals Versus the Order of the Data 6.0 0.5
y
4.5 l
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a
n 0.0
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q 3.0
e
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r R F -0.5 1.5 0.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 1 5 10 15 20 25 30 Residual Observation Order Figure 4.4 Residual plot for F. velutipes supplemented with IAA and BAP
Contour and surface plots were constructed using MINITAB® 14 to evaluate the response (mycelia growth rate) when two different parameters (BAP and IAA) were varied simultaneously. Based on Figure 4.5 and Figure 4.6, it indicated that the plots represent a saddle response surface. As the colours showed in contour plot gets darker, the response increases. The highest peak for mycelial growth rate was obtained when both of the growth hormone concentrations were at low level. From the stationary point near the center of the plots, simultaneously increasing or decreasing both concentrations of hormones leads to a decrease in the growth rate of mycelial. Both contour and surface plots were based on a regression model. Hence, to obtain optimum growth of F. velutipes mycelium, lower concentration of BAP and IAA are recommended to be used.
43
Contour Plot of Growth rate vs IAA, BAP 1.50 Growth rate < 9.8 9.8 - 10.0 10.0 - 10.2 1.25 10.2 - 10.4 10.4 - 10.6 > 10.6
A 1.00
A
I
0.75
0.50 0.50 0.75 1.00 1.25 1.50 BAP
Figure 4.5 Contour plot of mycelial growth rate versus plant growth hormones Growth hormones used are 6-benzylaminopurine (BAP) and β-indole acetic acid (IAA)
Surface Plot of Growth rate vs IAA, BAP
10.5
Growth rate 10.2 9.9 1.5 9.6 1.0 IAA 0.5 1.0 0.5 BAP 1.5
Figure 4.6Surface plot of mycelial growth rate versus plant growth hormones Growth hormones used are 6-benzylaminopurine (BAP) and β-indole acetic acid (IAA)
The response optimizer in MINITAB® 14 software was used to identify the combination of plant growth hormones concentration that jointly optimise the growth rate of mycelial. Based on the result obtained from CCD, the lower and upper target values were key-in as 9.28 mm/day and 10.64 mm/day, respectively. The optimal value
44 obtained was shown in Table 4.7. The maximum attribute response was showed 10.53 mm/day with a desirability of 0.94764. It indicates that for 100 times experiment runs, it is possible to achieve 95 times the targeted growth rate of F. velutipes mycelium. To obtain the maximum response (10.53 mm/day), MEA needs to be supplemented with the combination of 0.5 mg/L BAP and 0.5 mg/L IAA.
Table 4.7 Predicted value of mycelial growth rate (mm/day) at optimum concentration of growth hormones Global solution Predicted responses BAP IAA Mycelia growth rate Composite Desirability (mg/L) (mg/L) (mm/day) desirability 0.5 0.5 10.53 0.94764 0.94764 Growth hormones used are 6-benzylaminopurine (BAP) and β-indole acetic acid (IAA)
4.1.2 Verification
The predicted value above was then verified experimentally in order to verify the adequacy of final reduced models. With the predicted optimum hormone concentrations, the experimental mycelial growth rate value obtained was 10.53±0.27 mm/day (refer to Appendix C). Hence, there was no significant difference between experimental and predicted values. The concentration of 0.5 mg/L BAP and 0.5 mg/L
IAA as chosen to be as supplemented in MEA for growing F. velutipes mycelium as inoculum.
4.2 Selection of Carbon-Source consisting of Agroresidues used in Fruiting
Substrate Formulation for F. velutipes Cultivation
The effect of different agroresidues (SD, PS, EFB and PPF) in single and combination formulations on the radial mycelial growth rate was determined. The growth of mycelium was estimated by measuring of the linear growth of hyphae on a plate where growth occurs as a linear function of time (day). In Table 4.8, for single substrates, PPF (100) and EFB (100) showed higher mean radial growth rates of mycelium of 6.64±0.40 and 6.17±0.39 mm/day respectively. PS (100) showed the
45 lowest rate of 4.60±0.09 mm/day. However, when PS was combined with other agroresidues, the growth rate of mycelium increased slightly. The combination of SD with other agroresidues also showed higher mycelial growth rates compared to SD alone
(5.12±0.32 mm/day). Among the combined agroresidues, SD+PPF (75:25), PS+PPF
(50:50) and SD+PS (50:50) showed higher growth rates, 7.20±0.02, 6.84±0.32 and
6.78±0.49 mm/day with C:N 102.41, 124.35 and 101.66, respectively. The C:N of substrate was calculated by dividing the estimated carbon-content with the nitrogen- content. Based on the C:N ratio, EFB (100) showed the highest C:N ratio of 249.19 while the lowest was SD (100) of 94.72. From the Pearson correlation analysis, there was very weak negative correlation (-0.005) shown between C:N ratio with mycelial growth rate (p-value = 0.971). This shows that the C:N ratio of substrates did not affect the growth rate of F. velutipes mycelium. The C:N ratio of substrate is not the only factor affecting mycelium growth rate. The size of water activity particle substrates may also affect the mycelial growth rate. Among the substrate tested in this study, PPF and
SD were the finest size of particle compared to fibrous EFB and chopped PS.
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Table 4.8 Effect of various carbon-source agroresidues on the radial mycelia growth rate of F. velutipes (mm/day) Substrate Ratio mixtures by C:N Mycelial growth rate formulation weight (%) ratio (mm/day) SD 100 94.72 5.11±0.32a PS 100 110.57 4.60±0.09b EFB 100 249.19 6.17±0.39cd PPF 100 140.42 6.64±0.40d SD+PS 75:25 95.34 6.70±0.06d 50:50 101.66 6.78±0.49de
25:75 105.81 5.88±0.12cd
SD+EFB 75:25 115.16 6.70±0.09de 50:50 138.87 6.72±0.04de
25:75 177.18 6.35±0.08d
SD+PPF 75:25 102.41 7.20±0.02e 50:50 113.00 6.88±0.03de
25:75 123.90 6.27±0.06d
PS+EFB 75:25 129.81 5.06±0.26a 50:50 157.66 5.06±0.76a
25:75 192.51 6.13±0.09cd
PS+PPF 75:25 116.34 6.33±0.07d 50:50 124.35 6.84±0.32de
25:75 131.02 6.47±0.04d
EFB+PPF 75:25 210.36 6.46±0.11cd 50:50 181.21 6.26±0.16cd
25:75 158.56 6.64±0.14de Each value is expressed as mean±standard deviation of five replicates. The same letters denotes insignificant statistical differences (p≤0.05). Materials used as are sawdust (SD), paddy straw (PS), empty fruit bunches (EFB) and palm pressed fiber (PPF).
The selection of the fruiting substrate with which to determine the effect of carbon-source on the production yield of basidiocarps (the formation of primordia can be seen in Figure 4.8, and the fresh basidiocarp of F. velutipes is as shown in Figure
4.9) and BE was based on mycelial growth rate. Therefore, the substrate with the highest mycelial growth rate from each combination of two types of agroresidues was selected. The substrates were SD+EFB (50:50), SD+PS (50:50), PS+EFB (25:75),
SD+PPF (75:25), PS+PPF (50:50) and EFB+PPF (25:75). Single PPF (100) was also selected since it showed the highest mycelial growth rate compared with the other singular agroresidue substrates. The range of C:N ratio between those medium substrates was 100 – 195. This study was conducted by using polypropylene plastic
47 bags and the mycelial growth extension was measured. There was no adjustment done on the pH of substrates. The purpose was to observe whether the natural pH of substrate will affect the growth of mycelium. Based on Table 4.9, PPF (100) showed the lowest pH of 4.71 while the highest was 6.76 for SD+PS (50:50). These substrates PPF (100) and EFB+PPF (25:75), having low pH of 4.71 and 5.06, respectively, showed dense mycelial growth
In Table 4.9, the highest mycelial growth rate was obtained using SD+PS
(50:50), SD+PPF (75:25) and PPF (100) at 2.05±0.11, 1.90±0.60 and 1.79±0.13 mm/day, respectively. The lowest were PS+EFB (25:75) and PS+PPF (50:50) showing growth rates of 1.26±0.07 and 1.25±0.17 mm/day, respectively. Even though SD+PS
(50:50) showed the highest growth rate of mycelial, the average total yield of basidiocarps was among the lowest (41.21±12.20 g per bag). The PPF (100) was one of the formulations with dense mycelium (as shown in Figure 4.7), and gave the highest average total yield of basidiocarps (85.93±6.47 g per bag) and B.E. (129.06±14.51%).
The combination of SD+PPF (75:25) showed sparse mycelium thickness, with lower average total basidiocarp weight of 32.08±5.55 g per bag and BE of 74.41±9.62%. The combination of EFB+PPF (25:75) showed dense mycelium thickness and BE of
112.00±9.12%, but low average total yield of mushroom production of 41.29±35.87 g per bag.
The results obtained suggest that all the lignocellulosic agroresidues tested have good potential to be used as the growth substrates of F. velutipes. PPF showed the most efficient substrate to be used as fruiting substrate.
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Table 4.9 Effect of selected fruiting substrate formulations on the mycelial growth rate (mm/day), mycelium thickness, yield of F. velutipes basidiocarp (g) and biological efficiency, BE (%) Time for Average Average dry Average Average total Substrate complete mycelial Average biological Average pH weight of Mycelium basidiocarp yield formulations (%) spawn run growth rate efficiency (%) substrate (g) thickness (g per bag) (days) (mm/day) SD+EFB (50:50) 6.33±0.04a 45.96±1.76a 49 1.76±0.13b dense 57.91±19.59a 125.27±39.68a SD+PS (50:50) 6.76±0.03b 33.27±0.82b 44-45 2.05±0.11bc sparse 41.21±12.20ab 123.91±7.07a PS+EFB (25:75) 6.42±0.03c 35.00±2.28b 50 1.26±0.07a dense 65.08±15.24ac 185.09±36.98ab SD+PPF (75:25) 5.54±0.03d 42.93±2.12ac 31-45 1.90±0.60b sparse 32.08±5.55ab 74.41±9.62ac PS+PPF (50:50) 5.33±0.06e 39.54±2.94c 50 1.25±0.17a sparse 59.02±18.17a 150.89±50.35ab EFB+PPF (25:75) 5.06±0.02f 55.24±0.71d 49 1.63±0.07ab dense 41.29±35.87ab 112.00±9.12ac PPF (100) 4.71±0.01g 66.79±2.97e 49 1.79±0.07ab dense 85.93±6.47c 129.06±14.51a
Each value is expressed as mean±standard deviation of three replicates. Materials used are sawdust (SD), paddy straw (PS), empty fruit bunches (EFB) and palm press fiber (PPF).The same letters denotes insignificant statistical differences (p≤0.05).
49
Figure 4.7 Mycelium thickness (From left; sparse, and dense).
Figure 4.8 Primordia formation on the surface at the top of a fruiting bag.
50
Figure 4.9 Fresh F. velutipes basidiocarps after harvest.
4.3 Effect of Supplementation of Nitrogen-source on Mycelial Growth and
Yield of F. velutipes
In this study supplementation of RB and SY as nitrogen sources was investigated to determine whether growth and yield would be enhanced. Previously, this study has determined that PS+EFB (25:75), PS+PPF (50:50) and PPF (100) supported highest BE, and hence were selected as fruiting substrate formulations for the cultivation of F. velutipes. As for the control in this study, the industrial commercialized combination of SD+RB (80:20) was used. Table 4.10 shows that the supplementation by
RB and SY at concentration range of 5.0 – 20.0% lowered the C:N ratio of each substrate formulation.
The fruiting substrate, PS+EFB (25:75), supplemented with RB+SY (5.0:5.0) giving C:N ratio of 142.6 exhibited the highest mean mycelial growth rate of 2.39±0.18 mm/day. Based on ANOVA analysis as displayed in Table 4.10, there was significant difference in mycelial growth rate between nitrogen supplemented and their respective unsupplemented formulations except for PPF(100) supplemented with RB+SY at
51
(5.0:5.0) and (20.0:5.0) concentrations. This was supported by their p-value was greater than 0.05 (refer to Appendix D).
The unsupplemented fruiting substrate, PS+EFB (25:75) with C:N ratio of 192.5 gave the highest percentage BE of 185.09 ± 36.98%. Similarly, the percentages of BE from the all the nitrogen supplemented formulations were lower than the non- supplemented formulations. However, the range of BE of both supplemented and non- supplemented formulations were higher than the control formulation consisting of SD supplemented with 20% RB.
In conclusion, the growth rate of F. velutipes mycelium was increased in nitrogen supplemented formulations compared with the unsupplemented formulations.
For all the formulations, however, the supplementation of nitrogen source in the fruiting substrates lowered the percentages of BE. Therefore, lower C:N ratio enhances the growth of vegetative phase of F. velutipes but not the fruiting yield (basidiocarp formation).
52
Table 4.10 Effect of nitrogen supplements on the average mycelial growth rate (mm/day), basidiocarp yield (g) and biological efficiency (%) of F. velutipes N-source Average dry Mycelia Mycelia Fruiting substrate supplements (%) C:N weight of growth rate Total yield (g) BE (%) thickness RB SY substrate (g) (mm/day) SD (100) 20.0 0.0 75.0 50.81±6.96 1.80±0.12 sparse 39.80±14.39 77.47±20.19 PS + EFB (25:75) 0.0 0.0 192.5 35.00±2.28a 1.26±0.07a dense 65.08±15.24b 185.09±36.98b 20.0 20.0 86.7 44.94±2.98b 2.00±0.03b dense 41.38±7.52ab 92.33±18.37a
5.0 5.0 142.6 43.15±1.33b 2.39±0.18c dense 27.81±9.20a 65.91±22.59a
12.5 12.5 105.8 40.45±0.63b 2.06±0.08b dense 54.45±15.57ab 134.78±39.28ab
20.0 5.0 110.2 56.29±3.47c 1.87±0.26b dense 73.88±18.89b 129.47±33.60ab
5.0 20.0 101.4 44.49±0.59b 2.12±0.05b dense 27.43±18.50a 61.43±40.98a PS + PPF (50:50) 0.0 0.0 124.4 55.24±0.71a 1.25±0.17a sparse 59.02±18.17ab 150.89±50.35b 20.0 20.0 76.8 58.11±2.73b 1.69±0.03b dense 77.63±18.16b 132.90±26.51ab
5.0 5.0 118.0 47.35±5.24ab 1.64±0.14b dense 72.90±7.41b 147.54±23.13b
12.5 12.5 91.4 45.76±11.60ab 1.72±0.22b dense 58.33±27.95ab 138.35±81.27ab
20.0 5.0 95.4 50.36±1.00ab 2.13±0.03c dense 24.02±2.52a 48.10±5.93a
5.0 20.0 87.4 49.52±8.05ab 1.67±0.11b dense 74.16±14.56b 137.47±26.21ab PPF (100) 0.0 0.0 140.4 66.79±2.97ab 1.79±0.07a dense 85.93±6.47c 129.06±14.51a 20.0 20.0 75.4 73.13±0.58a 2.17±0.50b dense 55.36±19.12ab 75.88±27.24bc
5.0 5.0 112.0 61.38±1.85b 1.99±0.03ab dense 46.38±11.43a 75.43±17.38bc
12.5 12.5 88.7 62.34±1.92b 2.16±0.02b dense 55.31±2.47ab 88.75±3.66bc
20.0 5.0 92.5 73.10±4.93ab 2.02±0.13ab dense 69.32±2.54bc 95.82±7.62b
5.0 20.0 84.9 66.77±2.44ab 2.22±0.09b dense 43.84±3.23a 65.33±5.02c Each value is the mean±standard deviation of five replicates. The same letters denote insignificant statistical differences (p≤0.05). Materials used were paddy straw (PS), empty fruit bunches (EFB), palm-pressed fibre (PPF), rice bran (RB), and spent yeast (SY).
53
By using experimental factorial design, mathematical method was set to analyse the effects of supplementation of nitrogen-source in the fruiting substrate on F. velutipes mycelium growth rate. The analyses were performed for PS+EFB (25:75), PS+PPF
(50:50) and PPF (100) formulations, separately. The experimental data for each formulation were fitted to a second-order polynomial (Equation 4.1). The model consisted of main and interaction terms for a single factor and between two different factors effect, respectively. The main effect was calculated as the mean change in mycelial growth rate when the concentration of one of the supplements was modified from low to high percentage. A Pareto chart of the effect of mycelial growth rate was used to compare the relative magnitude and the statistical significance of both main (RB and SY) and interaction (RB*SY) effects, at the significance level, α = 0.05. A main effect plot was also used to show the consistent difference between the concentrations of a factor.
4.3.1 Analysis of effect nitrogen-source supplementation for PS+EFB (25:75) as main carbon-source
Table 4.11 showed that the main effects of RB (0.003) and the interaction of
RB*SY (0.039) were significant on the mycelial growth rate which their p-values were less than 0.05. But there was no significant effect shown by the main SY (0.427). The coefficient of determination, R2 was given as 0.6781, indicating a poor correlation between experimentally observed and predicted values. The adjusted R2 was given as
0.5493. This indicates the percentage of goodness fit between the experimental and the predicted results. This was supported by low value of standard deviation of error, S
(0.1463). The result of ANOVA was tabulated in Table 4.12, which showed that the p- value for the set of two-way interaction (0.039) was less than 0.05. Therefore, evidence exists of a significant interaction effect i.e. the effect of one factor depends on the level of another factor. The p-value for the set of main effects (0.010) was less than 0.05,
54
shows that evidence exists of a significant effect; at least one coefficient is not equal to zero.
Table 4.11 PS+EFB (25:75): Estimated effects of nitrogen-sources supplementation, coefficients, t-value and p-value for mycelial growth rate (mm/day). Coefficient's Term Effect Coefficient T P Standard Error Constant 2.0933 0.04224 49.55 0.000
RB -0.3233 -0.1617 0.04224 -3.83 0.003 SY -0.0700 -0.0350 0.04224 -0.83 0.427 RB*SY 0.2000 0.1000 0.04224 2.37 0.039 Center point -0.0333 0.09446 -0.35 0.731
S = 0.146333 R2 = 67.81% R2(adjusted) = 54.93% Materials used are rice bran (RB) and spent yeast (SY).
Table 4.12 PS+EFB (25:75): Analysis of variance (ANOVA) for mycelial growth rate (mm/day) on supplementation substrates. Source DF Seq SS Adj SS Adj MS F P Main Effects 2 0.328333 0.328333 0.164167 7.67 0.010 2-Way Interactions 1 0.120000 0.120000 0.120000 5.60 0.039 Curvature 1 0.002667 0.002667 0.002667 0.12 0.731 Residual Error 10 0.214133 0.214133 0.214133
Pure Error 10 0.214133 0.214133 0.214133
Total 14 0.665133 where DF = degrees of freedom Seq SS = sequential sum of squares Adj SS = adjusted sum of squares Adj MS = adjusted mean squares
Based on Figure 4.10a, the normal probability plot was not too far from straight line. It seems that the normality assumption was to be satisfied for this data. From the graph residual versus the fitted values (Figure 4.10b), the data were homogenized. Most of the residuals lie within the range of 0.2 and -0.2, which indicates that there was not much different between the residuals result (experimental) and the model results
(predicted). The histogram of the residual (Figure 4.10c) proved that the data were normally distributed 0.0, which shows symmetric and bell-shaped. Another graph, the residual versus the order of the data (Figure 4.10d) suggested that there was outlier at observation order 2 with residual -0.297 (as mentioned in the statistical analysis,
Appendix D).
55
Residual Plots for Growth rate (mm/day) (a) (b) Normal Probability Plot of the Residuals Residuals Versus the Fitted Values 99 0.2 90
l
t
a
n
u 0.0
e
d
c 50 i
r
s
e
e
P
R 10 -0.2
1 -0.30 -0.15 0.00 0.15 0.30 1.80 1.95 2.10 2.25 2.40 Residual Fitted Value (c) Histogram of the Residuals (d) Residuals Versus the Order of the Data 4 0.2
y 3
l
c
a
n
u 0.0 e
d
u i
2 s
q
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e
r
R
F 1 -0.2
0 -0.3 -0.2 -0.1 0.0 0.1 0.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Residual Observation Order Figure 4.10 Residual plots for mycelia growth rate (mm/day) of F. velutipes on PS+EFB (25:75) supplemented with different concentrations of RB and SY.
Figure 4.11 show that the effect of supplementation of singular RB had highly significant effect on the F. velutipes mycelium growth rate. The combination of RB*SY however, was only slightly significant. In addition, the supplementation of singular SY showed no significant effect as the parameter did not passed the significant level line of
95% at 2.23. From this chart, RB was the preferable supplement for the fruiting substrate, PS+EFB (25:75). A main effect is an outcome plot that shows consistent difference between levels of a factor. From Figure 4.12, the line of RB shows that there was stronger effect on the greater slope of line. This indicates that the response mean
(mycelium growth rate) changed depending on the factor level (concentration of RB).
The 5% concentration of RB supplementation shows greater mean of mycelium growth rate (2.26 mm/day), while 20% of RB shows lower (1.93 mm/day). The line of SY showed almost horizontal (parallel to x-axis), which indicates that there was no or slight effect of the concentration of SY on mycelia growth rate. Based on Figure 4.11 and
Figure 4.12, the supplementation of 5% RB was chosen for the study of the effect on F. velutipes mycelia growth rate. The verification results, 1.71±0.05 mm/day (refer to
Appendix C), shown that the supplementation of 5% RB was not significant effect with 56
the others supplemented substrates. Hence, the percentages concentration of RB and
SY, either singular or combination, did not give any significant effect on F. velutipes mycelium growth rate.
Pareto Chart of the Standardized Effects (response is Growth rate (mm/day), Alpha = .05) 2.228 F actor Name A RB B SY A
m r AB
e
T
B
0 1 2 3 4 Standardized Effect
Figure 4.11 Pareto chart of standardized effects for mycelia growth rate (mm/day) of F. velutipes on PS+EFB (25:75) supplemented with different concentrations RB and SY. Materials used are rice bran (RB) and spent yeast (SY).
Main Effects Plot (data means) for Growth rate (mm/day)
RB SY Point Type 2.3 Corner Center
)
y
a
d / 2.2
m
m
(
e
t
a
r
h
t 2.1
w
o
r
G
f
o
n 2.0 a
e
M
1.9 5.0 12.5 20.0 5.0 12.5 20.0 Concentration (%) Figure 4.12 Main effects plot (data means) for mycelia growth rate (mm/day) of F. velutipes on PS+PPF (25:75) supplemented with different concentrations RB and SY. Materials used as are rice bran (RB) and spent yeast (SY).
57
4.3.2 Analysis of effect nitrogen-source supplementation for PS+PPF (50:50) as main carbon-source
` Table 4.13 shows that the main effects for RB (0.006), SY (0.020), and the interaction of RB*SY (0.011) showed significant effect on the mycelial growth rate with p-values were less than 0.05. The standard deviation of error, S, was given as
0.128, which indicates that a moderate degree of error during the experiment. The R2 and adjusted R2 were given as 0.7491 and 0.6471 respectively, indicates that poor correlation between experimentally observed and predicted values. Table 4.14 showed that the p-value for the set of two-way interaction (0.011) was less than 0.05. Therefore, evidence exists of a significant interaction effect that the effect of one factor depends on the level of another factor. The p-value for the set of main effects (0.004) was less than
0.05, shows that evidence exists of a significant effect; at least one coefficient is not equal to zero.
Table 4.13 PS+PPF (50:50): Estimated effects of nitrogen-sources supplementation, coefficients, t-value and p-value for mycelial growth rate (mm/day). Coefficient's Term Effect Coefficient T P Standard Error Constant 1.7817 0.03693 48.24 0.000
RB 0.2567 0.1283 0.03693 3.47 0.006 SY -0.2033 -0.1017 0.03693 -2.75 0.020 RB*SY -0.23 -0.1150 0.03693 -3.11 0.011 Center point -0.0583 0.08258 -0.71 0.496
S = 0.127932 R2 = 74.91% R2(adjusted) = 64.87% Materials used are rice bran (RB) and spent yeast (SY).
58
Table 4.14 PS+PPF (50:50): Analysis of variance (ANOVA) for mycelial growth rate (mm/day) on supplementation substrates. Source DF Seq SS Adj SS Adj MS F P Main Effects 2 0.321667 0.321667 0.160833 9.83 0.004 2-Way Interactions 1 0.1587 0.1587 0.1587 9.7 0.011 Curvature 1 0.008167 0.008167 0.008167 0.5 0.496 Residual Error 10 0.163667 0.163667 0.163667
Pure Error 10 0.163667 0.163667 0.163667
Total 14 0.6522 where DF = degrees of freedom Seq SS = sequential sum of squares Adj SS = adjusted sum of squares Adj MS = adjusted mean squares
Based on Figure 4.13a, the normal probability plots of residuals showed a straight line. It seems that the normality assumption was to be satisfied for this data.
From Figure 4.13b, the data were homogenized. Most of the residuals lie within the range of 0.10 and -0.10, which indicates that there was not much different between the residuals result (experimental) and the model result (predicted). This was supported by
Figure 4.13c signifies that the data were normally distributed at 0.00, which shows symmetric and bell-shaped. Figure 4.13d suggested that there was outliers at observation order 12 with residual -0.243 (as mentioned in the Appendix D).
Residual Plots for Growth rate (mm/day) (a) (b) Normal Probability Plot of the Residuals Residuals Versus the Fitted Values
99 0.2
90 0.1
l
t
a
n
u
e
d 0.0
c 50 i
r
s
e
e
P R -0.1 10 -0.2 1 -0.2 -0.1 0.0 0.1 0.2 1.7 1.8 1.9 2.0 2.1 Residual Fitted Value (c) Histogram of the Residuals (d) Residuals Versus the Order of the Data 0.2 4.8 0.1
y
l
c 3.6
a
n
u
e
d 0.0
u i
s q 2.4
e
e
r R -0.1
F 1.2 -0.2 0.0 -0.2 -0.1 0.0 0.1 0.2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Residual Observation Order Figure 4.13 Residual plots for mycelial growth rate (mm/day) of F. velutipes on PS+PPF (50:50) supplemented with different concentrations of RB and SY.
59
Figure 4.14 shows that both main and interaction effects significant higher than the significance level line of 95% (α = 0.05) at 2.23. The RB showed highly significant effect compared to the interaction of RB*SY and SY because it extends the furthest.
From Figure 4.15 both RB and SY showed that there were stronger effects on the greater slopes of line. This indicates that the mean of mycelial growth rate was dependent on the percentage of supplement concentration. The 20% concentration of
RB supplementation shows greater mean of mycelial growth rate, while 5% of RB shows lower. The line of SY shows that the 5% concentration of SY supplementation shows greater mean of mycelial growth rate, while 20% of SY shows lower. Based on
Figure 4.14 and Figure 4.15, the supplementation of 20% RB was chosen for further study on the effect of F. velutipes mycelial growth rate. However, the experimental results, 1.61±0.06 mm/day (refer to Appendix C) shows that the supplementation of
20% RB was not significant effect with the other supplemented substrates. Hence, the percentages concentration of RB and SY, either singular or combination, did not give any significant effect on F. velutipes mycelia growth rate.
Pareto Chart of the Standardized Effects (response is Growth rate (mm/day), Alpha = .05) 2.228 F actor Name A RB B SY
A
m
r AB e
T
B
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Standardized Effect
Figure 4.14 Pareto chart of standardized effects for mycelia growth rate (mm/day) of F. velutipes on PS+PPF (50:50) supplemented with different concentrations RB and SY. Materials used as are rice bran (RB) and spent yeast (SY).
60
Main Effects Plot (data means) for Growth rate (mm/day) RB SY Point Type Corner 1.90 Center
)
y
a
d
/
m 1.85
m
(
e
t
a
r 1.80
h
t
w
o r 1.75 G
f
o
n
a
e 1.70 M
1.65
5.0 12.5 20.0 5.0 12.5 20.0 Concentration (%) Figure 4.15 Main effects plot (data means) for mycelial growth rate (mm/day) of F. velutipes on PS+PPF (50:50) supplemented with different concentrations RB and SY. Materials used as are rice bran (RB) and spent yeast (SY).
4.3.3 Analysis of effect nitrogen-source supplementation for PPF (100) as main carbon-source
From Table 4.15, the main effects for RB (0.971), SY (0.190) and the interaction between RB*SY (0.784) shows no significant effect on mycelial growth rate, that was, their p-values were greater than 0.05. From the statistical analysis obtained from
MINITAB® 14 (Table 4.15) showed that the standard deviation of error, S, was given as
0.236. This shows a moderate degree of error during the experiment. The coefficient of determination, R2, and the adjusted R2 were given as 0.1825, and 0.0000 respectively, indicates that no correlation between experimentally observed and predicted values.
Table 4.16 shows that the p-values for the set of two-way interaction (0.784) was greater than 0.05, which indicates that the two factors (RB and SY) were not dependable.
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Table 4.15 PPF (100): Estimated effects of nitrogen-sources supplementation, coefficients, T-value and P-value for mycelial growth rate (mm/day) Coefficient's Term Effect Coefficient T P Standard Error Constant 2.0992 0.06819 30.78 0.000
RB -0.0050 -0.0025 0.06819 -0.04 0.971 SY 0.1917 0.0958 0.06819 1.41 0.190 RB*SY -0.0383 -0.0192 0.06819 -0.28 0.784 Center point 0.0642 0.15249 0.42 0.683
S = 0.236234 R2 = 18.25% R2(adjusted) = 0.00% Materials used as are rice bran (RB) and spent yeast (SY).
Table 4.16 PPF (100): Analysis of variance (ANOVA) for mycelial growth rate (mm/day) on supplemented substrates. Source DF Seq SS Adj SS Adj MS F P Main Effects 2 0.110283 0.110283 0.055142 0.99 0.406 2-Way Interactions 1 0.004408 0.004408 0.004408 0.08 0.784 Curvature 1 0.009882 0.009882 0.009882 0.18 0.683 Residual Error 10 0.558067 0.558067 0.055807
Pure Error 10 0.558067 0.558067 0.055807
Total 14 0.682640 where DF = degrees of freedom Seq SS = sequential sum of squares Adj SS = adjusted sum of squares Adj MS = adjusted mean squares
Based on Figure 4.16a, the normal probability plots of residuals showed the straight line. It seems that the normality assumption was to be satisfied for this data.
From Figure 4.16b, the data were homogenized which most of the residuals fall within the range of 0.20 and -0.20, which indicates that there were not much difference between the residual result (experimental) and the model result (predicted). Residuals versus the fitted values were used to detect non-constant variance, missing higher-order terms, and outliers. In theory, the residuals should be scattered randomly around zero.
The histogram of the residual is used to detect multiple peaks, outliers and non- normality. Figure 4.16c signifies that the data were normally distributed at 0.00, which shows symmetric and bell-shaped. Another graph, the residual versus the order of the data (Figure 4.16d) suggested that there are outliers at observation order 1 with residual
0.577 (as mentioned in Appendix D). Figure 4.16d displays a non-clear pattern
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indicating time did not affect the result of the experiment and the data obtained. Figure
4.17 shows that supplementation of nitrogen-source was not significant as the parameters (RB, SY, and RB*SY) did not passed the significant level line of 95% at
2.23. Hence, the percentages concentration of RB and SY, either singular or combination, did not give any significant effect on F. velutipes mycelia growth rate.
Residual Plots for Growth rate (mm/day) (a) Normal Probability Plot of the Residuals (b) Residuals Versus the Fitted Values 99 0.6 90 0.4
l
t
a
n
u
e 0.2
d
c 50 i
r s
e e 0.0
P R 10 -0.2 1 -0.50 -0.25 0.00 0.25 0.50 2.00 2.05 2.10 2.15 2.20 Residual Fitted Value
(c) (d) Histogram of the Residuals Residuals Versus the Order of the Data 8 0.6
6 0.4
y
l
c
a
n
u
e 0.2
d u 4 i
s q
e e 0.0
r
R F 2 -0.2 0 -0.2 0.0 0.2 0.4 0.6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Residual Observation Order
Figure 4.16 Residual plots for mycelial growth rate (mm/day) of F. velutipes on PPF (100) supplemented with different concentrations of RB and SY.
Pareto Chart of the Standardized Effects (response is Growth rate (mm/day), Alpha = .05) 2.228 F actor Name A RB B SY B
m
r AB
e T
A
0.0 0.5 1.0 1.5 2.0 2.5 Standardized Effect
Figure 4.17 Pareto chart of standardized effects for mycelial growth rate (mm/day) of F. velutipes on PPF (100) supplemented with different concentrations RB and SY. Materials used are rice bran (RB) and spent yeast (SY). 63
CHAPTER 5.0 DISCUSSION
5.1 Effect of Growth Hormones on F. velutipes Mycelial Growth
In order to achieve reliable and vigorous fungal growth and high yield of mushroom production, good quality mycelium used as inoculums is vital. This study investigates the effect of growth hormones on the vegetative growth of F. velutipes for the preparation of inoculum. The growth hormones studied were BAP and IAA. BAP is a synthetic cytokinin, and IAA is an auxin then can be found naturally in plants.
Previous study by Mukhopadhyay et al. (2005) showed IAA and KIN enhanced growth and protein content of Pleurotus sajor-caju, however, there was no study conducted on the effect of any plant growth hormones on either vegetative or reproductive phase of F. velutipes. In this study, a set of experiments was conducted using statistical design of experiments (DOE) and developed by MINITAB® 14 software. DOE is an efficient technique of optimizing a process in experimentation to produce high quality products, economic, and ensures a stable and reliable process (Montgomery, 2001).
In a preliminary experiment, a full factorial design was used, which BAP and
IAA were chosen as the factors with low (1.0 mg/L) and high (10.0 mg/L) level of concentrations. By using a factorial design, each replication of the experiment all possible combinations of the levels of the factors are investigated. The supplementation
MEA of BAP and IAA were found to enhance mycelia growth rate of F. velutipes significantly (p≤ 0.05) (Table 4.1). Combination of IAA and BAP with ratio 1:1 at low level of concentration (1.0 mg/L) showed the highest effect compared with those singular effects. Zhong et al. (1998) also reported that the highest production of polysaccharide (1.97 g/L) was obtained with the combination of 10.0 mg/L napthalene acetic acid (NAA)+0.1 mg/L kinetin (KIN) in suspension cultures of Panax ginseng C.
A. Meyer. According to Reski (2007), a relatively low IAA concentration could stimulate tissue growth, while higher IAA concentrations were toxic in plant tissue
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culture. Guha and Banerjee (1974) reported that the stimulation of growth of Agaricus campestris by IAA at a concentration of 0.4 mg/L, however at higher concentration of
0.4 mg/L, the growth was inhibited. Chodchoi (1986) reported that with another type of auxin, the highest mycelium growth of Auricularia polytricha (Mont.) Sacc. with NAA at 10 ppm, and lowest with 100 ppm NAA. Mukhopadhyay et al. (2005) also reported that KIN, a synthetic cytokinin, stimulate highest mycelia growth rate of P. sajor-caju at low level concentration of 2.0 mg/L, and inhibition of growth was noted when the hormone was used at a concentration above 5.0 mg/L. Thus, lower concentration of IAA and BAP with ratio 1:1 was used for this study.
Further, the optimization of the concentrations of IAA and BAP using
MINITAB®14 software was performed. The response surface method (RSM) was used to analyze the central composite design (CCD). RSM is a collection of mathematical and statistical techniques that are useful for the modeling and analysis of problems in which a response of interest is influenced by several variables and the objectives is to optimize this response (Montgomery, 2001). CCD was used to fit a second-order model, as 2 x 2 factorial has been used to fit a first-order model. Growth hormone was set at the concentration range of 1.5–0.5 mg/L. The maximal mycelial growth rate obtained was
10.53±0.27 mm/day with the supplementation of 0.5 mg/L IAA+0.5 mg/L BAP. The lowest growth of 9.40±0.15 mm/day was obtained when 1.5 mg/L IAA+1.0 mg/L BAP was used. The concentration of 0.5 mg/L BAP+0.5 mg/L IAA was chosen as supplemention for MEA media in the preparation of inoculum of F. velutipes. Similar study by Dey et al. (2007) on the effect of IAA on mycelium growth of Ganoderma lucidium (Curtis.) P. Karst obtained at low concentration 5.0 mg/L. Mukhopadhyay et al. (2005) in their study found that plant growth regulators viz. IAA, gibberellic acid
(GA3), and kinetin (KIN) at different concentration increased the biomass production of
P. sajor-caju by 15 – 26% and also increased in protein content of the mycelia. Paul et
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al. (1994) also reported that IAA, GA3 and KIN at an optimum concentration increased the biomass production of food yeast Kluyveromyces fragilis grown in deproteinized whey, but there was no effect of hormones on the protein content of biomass.
Indole acetic acid had been used to accelerate the elongation and increase the growth and the divisions of plant cells (Zhao, 2010). Probably, auxins also controlled the fungal cell elongation (Yanagishima, 1963) and differentiation (Tomita et al., 1984).
Alexander and Lippert (1989) reported in their study that IAA at 0.05 ppm concentration enhanced best mycelia proliferation of Calvatia gigantean (Batsch. ex
Pers.) Lloyd, Calvatia booniana A. H. Sm. and Calvatia craniiformis (Schwein.) Fr..
Maniruzzaman (2004) in his study found the best mycelium growth at 5 ppm IAA gave the highest yield in L. edodes mushroom production. Dey et al. (2007) also reported that the MEA medium supplemented with 5.0 mg/L IAA gave the highest mycelium growth
(8.20 cm) of Calocybe indica (Purkay. & A. Chandra) mushroom, and lowest (6.50 cm) with 20.0 mg/L. Cytokinins were effective compounds in the regulation of the growth of plants by increasing the division of cells (Korkutal et al., 2008). 6-Benzylaminopurine
(BAP), or known as benzyl adenine, is a first-generation synthetic cytokinin that promotes plant growth and development responses, setting blossoms and stimulating fruit richness by stimulating cell division. KIN, a synthetic cytokinin, was reported increasing the biomass and protein content of A. bisporus (Guha and Banerjee, 1974), sequently on yeast K. fragilis (Paul et al., 1994), and followed with P. sajor caju
(Mukhopadhyay et al., 2005).
5.2 Selection of Agroresidues as Carbon-Source in The Formulations of Fruiting
Substrate for F. velutipes
Generally, mushroom growth requires carbon, nitrogen and inorganic compounds as its nutritional source, and the main carbon source is derived from cellulose, hemicelluloses and lignin. Sawdust was commonly used as the fruiting 66
substrate in mushroom production. However, the low availability of sawdust has become a serious problem to the mushroom growers. Thus, the potential of alternative substrates need to be investigated to vary the fruiting substrate besides using purely sawdust. Since paddy straw, EFB and PPF are produced abundantly in Malaysia, they were selected to evaluate the possibility of using them as fruiting substrates either individually or in combination. The availability and low cost of these agroresidues is an important consideration for growers in making a high profit by lowering the cost of materials. Most of these lignocellulosic agroresidues were disposed of through incineration and dumping. Thus, from the environmental aspect, recycling these agroresidues would be a great effort to reduce the cause of environmental pollution.
By referring to the analyses done in Appendix B, paddy straw (PS), EFB and
PPF contained 77.40% carbon and 0.70% nitrogen, 89.71% carbon and 0.36% nitrogen, and 84.25% carbon and 0.60% nitrogen, respectively. Sawdust (SD) contains 85.25% carbon and 0.90% nitrogen. This shows that the carbon and nitrogen percentage of PS,
EFB and PPF were on par to that SD, indicating that these agroresidues have great potential to be used as a fruiting substrate for F. velutipes. In this study, these SD, PS,
EFB and PPF were tested, singularly and in combination with other substrates for the growth of F. velutipes mycelium. Mycelium growth showed that F. velutipes has the ability to degrade all lignocellulosic formulations agroresidues and support growth
(Table 4.8). No study has been conducted using these agroresidues as a fruiting substrate for F. velutipes production. However, PS has been used as the carbon-source for Pleurotus sp. (Madan et al. 1987; Bisaria et al. 1987) and Agaricus sp. (Ho and
Peng 2006). EFB (Muhammad et al. 2008) and PPF (Klitsaneepaiboon and Bunkong
1990) has been used as fruiting substrates for P. sajor-caju, and Abd Razak et al. (2012) also reported that EFB and PPF are potential fruiting substrate for Auricularia polytrichia.
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However, there exist variations in mycelial growth rate on different substrates and thus might be due to variations in the chemical composition and C:N ratios of the substrates. The range of C:N ratio of formulated substrates used in this study was 94 –
250, producing a mycelial growth rate in the range of 4.6 – 7.2 mm/day (Table 4.8). The formulated substrates with a range of C:N between 100 – 125 showed higher mycelial growth rates ranging from 6.8 - 7.0 mm/day. However, there was a very weak negative correlation between the C:N ratio and the mycelia growth rate shown in Table 4.8. As low C:N ratio of substrates, the mycelial growth rate showed higher. Philippoussis et al.
(2001) also reported a positive correlation between mycelial growth and low C:N ratio of substrates in the cultivation of Pleurotus spp. Generally, Charlesworth (1995) estimated that C:N ratio range of 10 – 80 is suitable for mushroom fruiting substrate media. Yung and Ho (1979) reported that the optimum C:N ratio for V. volvacea is about 75 - 80, but that ratios between 32 - 150 are almost as effective. Plants material with low C:N ratios were degraded more rapidly than those with high C:N ratios, indicating that mycelial growth rate is related to the bioavailability of nitrogen
(Philippoussis et al., 2003; Carreiro et al., 2000). As mentioned previously, carbon is the main nutrient needed, especially during vegetative growth. Carbon sources provide the structural and energy requirement for the fungal cells (Chang and Miles, 2004).
The best substrate formulations that supported highest mycelial growth viz
SD+EFB (50:50), SD+PS (50:50), PS+EFB (25:75), SD+PPF (75:25), PS+PPF (50:50), and EFB+PPF (25:75) were further investigated for the yield of basidiocarp. This study was conducted using polypropylene plastic bags as the container. The combination of
SD+PS (50:50) showed the highest pH value (6.76) with the highest mycelia growth rate (2.05 mm/day). PPF (100) showed the lowest pH value (4.71), but the mycelia growth rate was among the highest (1.79 mm/day). Chang and Miles (2004) stated that
F. velutipes mycelium grew best at pH 4.0 – 8.0. Hence, all the pH value of the
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formulations tested were suitable for mycelium growth. Özçelik and Pekşen (2007) reported that the growth of Lentinula edodes mycelia was not affected by high pH value
(> pH 6).
With regard to nutrition, F. velutipes demonstrates biodegradation ability on lignocelluloses polymers, but the degree of benefit from them varies depending on carbohydrate content of by-product materials (Royse, 1985). The major component of lignocelluloses materials are cellulose, lignin and hemicellulose. Cellulose and hemicelluloses are macromolecules from different sugars, whereas lignin is an aromatic polymer synthesized from phenylpropanoid precursors (Pérez et al., 2002). The composition and percentages of these polymers vary from one plant species to another.
Tisdale et al. (2006), Palonen (2004), and Ward et al. (2000) reported that SD contains
37.7 - 49.5% cellulose, 10.7 - 25.0% hemicelluloses and 26.1 - 29.5% lignin. PS contains 22.8 – 38.4% cellulose, 17.7 – 28.5% hemicelluloses and 6.4 – 18.0% lignin
(Mata and Savoie, 2005; Howard et al., 2003; Obodai et al., 2003). EFB contained high range of 45 – 50% cellulose, with 25 - 35% hemicelluloses and 25 - 35% lignin (Khalil et al., 2007; Sreekala et al., 1997). PPF was reported by Astimar et al. (2002) to consist of 32.4% cellulose, 38.2% hemicelluloses and 20.5% lignin.
In Table 4.9, the combination of SD+PS (50:50) showed the highest mycelial growth rate (2.05 mm/day) on the polypropylene plastic bag. This might be due to the low percentage of lignin in PS; the low-molecular weight and soluble carbohydrates of this substrate are easily metabolised by mushroom mycelia (Kurt and Buyukalaca
2010). Hemicellulose is a polysaccharide with a lower molecular weight than cellulose.
The structural complexity of lignin, with its high molecular weight and insolubility make its degradation difficult (Pérez et al. 2002) as reflected by the lowest mycelial growth rate in PS+EFB (25:75). Chandhary et al. (1985) stated that there is an apparent correlation between the ability to degrade lignin and the production of phenolases,
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which oxidize phenolic compounds to simple aromatic compounds which can be absorbed by mushroom mycelium and uses for growth. The product of cellulolytic action in simple and soluble carbohydrates and the end products being glucose was absorbed by the fungal mycelium for growth and energy. Therefore cellulose rich substrates are good substrates for the cultivation of mushroom as it is easily being degraded (Quimio, 1987; Gerrits and Muller, 1965; Walksman and Nissan, 1932).
The differences of particle size of agroresidues used could contribute to the variation on the mycelia growth. In this study, the PS used was prepared was ground into 2 – 3 cm length, while SD, EFB and PPF were ground into finer particle. The larger size of PS led the lowest degradation. When PS combined with other substrates, the varying size of particle increased the rate of mycelia growth. PPF is observed to be much finer as compared to SD. Even though the particle size of ground EFB can be considered as fine, but it was also observed to be fibrous. When water was added, the finer substrates became more compact, thus reducing void available between residues.
The thin cell wall of PS may be less constrained than wood in adsorption of water thus resulting in higher moisture content. Han et al. (1981) determined that L. edodes, a wood-decaying fungus, obtains its nutrients from compounds in cell walls, but the time of cell wall breakdown by enzymes and the degree of enzyme destruction varied among tree species. The time taken is also influenced by the shiitake strain used, the substrate formula, and the amount of substrate available, the spawning rate, the spawn distribution and the temperature during incubation (Philippoussis et al., 2003; Royse and Bahler, 1986). Baysal et al. (2003) and Demirci (1998) reported that the slower spawn running may be due to excess nitrogen content, which is known to inhibit mycelia growth. The thickness of mycelia might be due to the high content of carbon in substrates.
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With regard to the yield of basidiocarp, it varied with the different substrates used (Table 4.9). There was a positive correlation (0.46) shown between the C:N ratio and total basidiocarp yield. Fruiting substrates with a high nitrogen content resulted in a decline in the basidiocarp yield. PPF (100) contains the least amount of nitrogen (0.6%) and gave a high basidiocarp yield (85.94 g). The combination of SD+PPF (75:25) showed a lower yield (32.08 g), with a C:N ratio of 102 Substrates contains organic nitrogen sources and low in free ammonium, since excess of nitrogen will inhibit the formation of basidiocarp. The bioavailability of nitrogen in fruiting substrates is one of the factors that regulate enzyme production by wood rotting basidiomycete. Different enzyme production to degrade the lignocellulosic substrates, different abilities to form basidiocarp. Similarly findings to this study, Philippoussis et al. (2003; 2001; 2000) found that the growth rates of Pleurotus eryngii (DC.) Quél. and L. edodes also showed positive correlation with the C:N ratio. In this study, there was a weak negative correlation (-0.353) between mycelial growth rate and basidiocarp yield (Table 4.9).
However, previous studies have shown that the time of complete mycelium run was positively correlated with rapid primordial initiation and high yield of basidiocarp
(Naraian et al., 2009; Baysal et al., 2003; Demirci, 1998). According to Chang and
Miles (2004), a nitrogen compound that gives good mycelium growth may not provide a high yield of basidiocarp. Furthermore, a high concentration of nitrogen in substrate encourages mycelium growth and decreases the formation of basidiocarp.
Mushroom substrate may be defined as a kind of lignocellulosic material that supports the growth, development and fruiting of mushroom. Royse and Bahler (1986) stated that BE was significantly affected by the interaction between genotype, spawn run time, and substrate formulation. BE was calculated as the percentage ratio of the fresh weight of harvested mushrooms over the weight of dry substrate at inoculation, which indicates the fructification ability of the fungus utilizing the substrates. The BE of
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F. velutipes varied with substrate formulations. Substrates PS+EFB (25:75) and
PS+PPF (50:50) with low mycelial growth rate, produced the highest BE (Table 4.9).
However, substrates SD+PPF (75:25) and EFB+PPF (25:75) which showed higher mycelial growth rate, produced lower BE (Table 4.9). The fastest mycelium run was on
SD+PS (50:50) formulation but this substrate produced 41.21±12.20 g of basidiocarp with a BE of 123.91% (Table 4.9). The highest yield of basidiocarp was achieved with
PPF (100) of 85.93±6.47 g with a BE of 129.06±14.51 %. In this study, however, all the substrates showed greater percentage of BE compared to that in previous studies that used different lignocellulosic agroresidues. Lu et al. (1989) reported that 88% of cotton seed husk with additives produced a BE of 98.6% of F. velutipes, whereas 89% of paddy straw with additives produced a BE of 50.9%. The production of F. velutipes on maize straw substrate showed 73% BE (Ji et al., 2001). On coffee husk as a substrate, the BE reached about 56% with two flushes after 40 days, whereas on spent-ground as a substrate, the BE reached 78% (Leifa et al., 2001).
Nutritional contents and characteristics of lignocellulosic agroresidues are important factors that affect the basidiocarp yield. As mentioned previously, fungi require simple nutritional as it is heterotrophic and absorptive. Hence, the amount content of carbon and nitrogen in fruiting substrate are important for the structural and energy requirement for the fungal cells. Even though mushroom species have the ability to degrade lignocellulosic residues, they exhibit differences in production of enzymes to degrade substrates, and thus, different abilities to grow and formation of basidiocarp on the substarate. This study revealed PS as the potential of lignocellulosic agroresidue as an alternative fruiting substrate. There are numbers of studies reported that PS gave the best yield of different Pleurotus species. The percentages of B.E. of P. sajor-caju on PS were reported to be in the range of 46.6 - 149.4% (Pala et al., 2012; Kurt and
Buyukalaca, 2010; Nageswaran et al., 2003; Ragunathan et al., 1996). PS also has been
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reported as substrate for Agaricus species (Ho and Peng, 2006), but Ashrafuzzaman et al. (2009) reported that there is no yield of L. edodes on PS. The similarity of lignocellulosic composition of PPF to PS makes them good substrates for mushroom cultivation such as Pleurotus spp. (Klitsaneepaiboon and Bunkong, 1990). Amal et al.
(2008) reported that the highest yield of Pleurotus ostreatus(Jacq.) P. Kumm. was observed using the combination of substrate SD+PPF (50:50), which was0.19 kg with a
BE of 11.3% and PPF (100) showed the lowest BE which of 4.3%. EFB was very fibrous compared to other lignocellulosic agroresidues. Amal et al. (2008) also reported that the combination of SD+EFB (50:50) as a substrate showed a higher BE (8.6%) compared to EFB (100), where there was no yield of basidiocarps. Volvariella sp. and
Ganoderma boninense Pat. have also been reported to be cultivated on the EFB
(Sudirman et al., 2011). Thus, all the lignocellulosic agroresidues investigated in this study showed great potential for use as an alternative carbon-based fruiting substrate for
F. velutipes.
5.3 Effect of Rice Bran and Spent Yeast as Supplementation of Nitrogen-sources for Mycelial Growth and Yield of F.velutipes
Nitrogen is an essential element for cellular functions for growth and various metabolic activities particularly protein and enzymes synthesis (Upadhyay et al., 2002).
According to Moda et al. (2005), supplementing the fruiting substrate with nitrogen sources is a common method to increase productivity, as evaluated by biological efficiency (BE). The most common supplements are grains or their derivatives, such as rice, wheat or oat bran, ground corn etc (Stamets, 1993). Many growers also utilize grape pumice from wineries and spent barley from breweries as supplements (Stamets,
1993). Biological nitrogen rich supplements are more recommended due to being easier to implement, less expensive and more environmentally friendly (Pant et al., 2006;
Banik and Nandi, 2004).
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With regard to the effects of RB and SY supplements on mycelial growth rate and the production of F. velutipes basidiocarp, three formulations [PPF (100), PS+EFB
(25:75), and PS+PPF (50:50)] were selected, based on their high BE (Table 4.9).Results showed the supplementation with the nitrogen-sources lowered the C:N ratio of the fruiting substrate (Table 4.10). The supplemented substrates showed increased rates of mycelial growth, and had significant effects on growth compared to the non- supplemented substrates. Philippoussis et al. (2001) reported that there have been a positive correlation of low C:N ratio values of substrates used between mycelial growth for cultivation of Pleurotus spp.
From the validation of a Pareto chart for each base carbon substrate, RB shows positive effect on mycelial growth rate as a potential supplement on PS+EFB (25:75)
(Figure 4.9) and PS+PPF (50:50) (Figure 4.12), but there was no significant effect of supplements on PPF (100) (Figure 4.15). From the validation of a main effect plot for each fruiting substrate, 5% concentration of RB supplementation on PS+EFB (25:75) shows greater mean of mycelial growth rate (2.26 mm/day). On PS+PPF (50:50), 20% of RB shows greater mean of mycelial growth rate (1.91 mm/day), and 5% of SY also gave greater rate (1.88 mm/day). Ji et al. (2001) also reported that the highest mycelial growth rate of F. velutipes on wheat straw supplemented with 5% of wheat bran.
Moonmoon et al. (2011) reported that the highest mycelial growth of L. edodes was observed when 20% RB was supplemented to SD fruiting substrate. Eira and Minhomi
(1996) reported that L. edodes can also be cultivated on baggase supplemented with
20% RB. According to Fasidi and Kadiri (1993), the increased productivity of Lentinula subnudus Berk. supplemented with 30% RB can be attributed to the carbohydrates, amino acids and mineral elements in this supplement.
Contrary to the mycelial growth rate above, this study showed a decrease in the yield of basidiocarps and BE upon supplementation of nitrogen-sources (Table 4.10). 74
Similarly Philippoussis et al. (2002) also reported reduced yield on P. eryngii mushroom yield. According to Chang and Miles (2004), a nitrogen compound that gives good mycelial growth may be suitable for fruiting. A high concentration of nitrogen encourages mycelia growth and decreases the formation of fruiting bodies. Rajaratnam and Bano (1988) found that although natural substrates, such as woods, have very low nitrogen content, the basidiocarps of Pleurotus spp. were successfully produced.
Upadhyay et al. (2002) reported that the best range of nitrogen content for Pleurotus spp. is 3 - 6%. Thus, an excess of nitrogen content might be one of the factor affecting the yield of basidiocarp. Tang et al. (2001) reported that PS supplemented with 10% of
RB gave F. velutipes 76.73% of BE. Upadhyay et al. (2002) reported that the supplementation of 10% RB in wheat straw increased 17.5% BE of Pleurotus sp. from control. Mamiro and Mamiro (2011) reported that the highest BE (64.5%) of P. ostraetus was observed on PS supplemented with 25% of RB, but the efficiency decreased when supplemented with 50% and 75% of RB. Alam et al. (2010) also reported that increasing the amount of supplement resulted in an increase in BE (77.8%) of Calocybe indica up to 40% of RB, and then the efficiency decreased again. Kurt and
Buyukalaca (2010) stated that the high nitrogen content resulted in the decline in yield, whereby the total mushroom weight was found to be negatively correlated to C:N ratio in the cultivation of P. ostraetus and P. sajor-caju grown on substrates supplemented with wheat bran.
The percentages of BE can also be increased by ensuring favourable environmental condition for F. velutipes cultivation. Besides nutritional and chemical factors, almost any environmental factors, such as temperature, humidity, aeration and light, affect fruiting yield (Chang and Miles, 2004). However, this study shows that supplementation of nitrogen-sources to fruiting substrate formulations are optional to the growers for the cultivation of F. velutipes. When supplements are necessary extra
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care is required to discourage contamination. Contamination is one of the reasons that led to the insufficient yield of basidiocarp. In this study, we observed that green mold
(Trichoderma sp.) easily developed easily on the supplemented substrate. Rinker and
Alm (1998) stated that even though supplementation can increase mushroom yield to
25%, but it was also known that the supplements can serve as a food source for competitor molds such as Trichoderma sp. According to Kiran and Jandaik (1989), wheat bran attracted contaminants especially in the case of sparsely colonized substrates such as sawdust and wood shavings. Yildiz et al. (2002) also reported that the substrate supplemented with 25% bran increased the risk of contamination. Hence, in the absence of starch-based supplements, the probability for green mold to establish in the substrate is very low.
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CHAPTER 6.0 CONCLUSION
Mushroom cultivation is an economically important biotech-industry in
Malaysia that is expanding each year. This industry involves bioconversion of lignocellulosic materials into food for human consumption. Due to the awareness of the nutritional and medicinal values of mushrooms have resulted in an increasing demand of mushrooms as food. Since the production of Flammulina velutipes by Malaysian growers is lacking, they are mostly imported from China, Taiwan and Korea.
In the commercial mushroom industry in Malaysia, sawdust is largely used as the fruiting substrate for the cultivation of F. velutipes. Since the major commodity in
Malaysia are oil palm and rice production, the abundance of the lignocellulosic agroresidues from those commodities were selected to investigate the possibility of utilising these residues as fruiting substrate. In addition, spawn quality determines the success of a mushroom industry. To obtain good quality spawn, mycelium vigour is very important. These facts showed that supplementation of growth hormones enhances growth rate, hence, shorten the period for vegetative growth (spawn running) of F. velutipes mycelium. The optimum concentration of the combination of growth hormones is 0.5 mg/L IAA+0.5 mg/L BAP significantly enhanced the mycelial growth rate of F. velutipes to 10 mm/day. This is the first result in the effect of growth hormone on mycelial growth of F. velutipes. Further studies are to be conducted analyse the effect of different concentration of IAA and BAP on the amino acid content in mycelium. The supplementation of the growth hormones would probably change the content of amino acid in mycelium whether it is beneficial or otherwise. The effect of the growth hormones on secretion of mycelium enzyme could also be conducted as it is important to note the ability of mycelium in degradation of complex fruiting substrate.
PPF (100) and EFB (100) as fruiting substrates supported the highest mycelial growth rate while PS+EFB (25:75), PS+PPF (50:50) and PPF (100) produced the 77
highest percentage BE. The findings showed that nitrogen-sources (RB and SY) supplementation in PS+EFB (25:75), PS+PPF (50:50) and PPF (100), increased mycelial growth rate but the yield of basidiocarps and BE were reduced. This indicates that high nitrogen content activates mycelial growth rate but inhibits the induction of basidiocarps. This will lower the cost of substrate preparation and hence will economically benefit the growers. However, further study is needed to analyse the nutritional content of basidiocarps on these optimum substrates since the nutrient composition may depend on the fruiting substrate composition. The analysis of heavy metals and toxic chemicals could also be done to ensure that the agroresidues are safe to be utilized as fruiting substrate. The information from these analysis would be useful support the use of SD, PS, PPF and EFB in the cultivation of F. velutipes. Further study is needed to optimize the environmental conditions such as temperature, humidity and light, that could improve the basidiocarp yield and BE of F. velutipes.
The long-term value and significance of this research lies in the potential to improve the bioconversion of the lignocellulosic agroresidues by F. velutipes, thus increasing BE and improving mushroom quality. Additionally, the information gained from one particular mushroom will be relevance to the cultivation of other species of mushroom. By using those potential lignocellulosic materials for the cultivation of F. velutipes, it is beneficial to the human welfare by increasing food supply, reducing production cost and reducing environmental pollution.
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APPENDIX A
1.0 Flow chart of preparation of Malt Extract Agar (MEA) media
Dissolved 20 grams of MEA in 400 mL of distilled water.
Autoclaved the prepared media for 20 minutes at 121 ˚C and 15 psi.
The sterilized media was poured into 90 x 15 mm disposable Petri dishes. This step was carried out in the laminar flow to avoid contamination.
The media was allowed to cool for 30 minutes to be solidified.
Figure 1.1 The procedure of preparation of Malt Extract Agar (MEA) media
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APPENDIX B: CHEMICAL COMPOSITION
Table 1.1 The percentages of carbon and nitrogen in tested samples Sample Carbon (C; %) Nitrogen (N; %) Sawdust (SD) 85.25 0.90 Paddy straw (PS) 77.40 0.70 Empty fruit bunches (EFB) 89.71 0.36 Palm pressed fiber (PPF) 84.25 0.60 Rice bran (RB) 80.92 2.00 Spent yeast (SY) 25.63 2.00 The percentages of carbon of samples were tested by using Furnace method. The Kjeldahl method was used for determined percentage of nitrogen.
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APPENDIX C: EXPERIMENTAL DATA
1.0 Effect of Plant Growth Hormones on Mycelia Growth of Flammulina velutipes on Malt Extract Agar (MEA)
Table 1.1The experimental data of control: MEA without addition of Plant Growth Hormones Diameter (mm) Growth rate Day 4 5 6 7 10 (mm/day) 1 30.0 39.0 47.0 56.5 79.0 7.89 Replicate 2 27.0 36.0 46.0 56.0 80.0 7.77 no. 3 30.0 39.5 47.0 56.0 78.0 7.84 Average 7.83 Standard deviation 0.06
Table 1.2The experimental data of screening:Growth rate of F. velutipes grown on MEA supplementes with different hormone concentration Standard Run Center Factors (mg/L) Response: Mycelia Blocks order order point BAP IAA growth rate (mm/day) 2 1 1 1 10.0 1.0 10.06 13 2 0 1 5.5 5.5 10.37 4 3 1 1 10.0 10.0 10.23 3 4 1 1 1.0 10.0 10.11 1 5 1 1 1.0 1.0 10.57 15 6 0 1 5.5 5.5 10.33 11 7 1 1 1.0 10.0 10.01 7 8 1 1 1.0 10.0 10.15 6 9 1 1 10.0 1.0 10.05 8 10 1 1 10.0 10.0 10.20 5 11 1 1 1.0 1.0 10.47 9 12 1 1 1.0 1.0 10.45 12 13 1 1 10.0 10.0 10.15 10 14 1 1 10.0 1.0 10.07 14 15 0 1 5.5 5.5 10.32 Plant growth hormones used as are 6-benzylaminopurine (BAP) and β-indole acetic acid (IAA).
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Table 1.3 The experimental data of optimization: Growth rate of F. velutipes grown on MEA supplementes with different hormone concentration Standard Run Type of Factors (mg/L) Response: Mycelia Blocks order order point BAP IAA growth rate (mm/day) 28 1 -1 1 1.5 1.0 9.352 6 2 -1 1 1.5 1.0 9.277 4 3 1 1 1.5 1.5 9.831 2 4 1 1 1.5 0.5 10.16 30 5 -1 1 1.0 1.5 9.941 24 6 1 1 1.5 0.5 9.941 13 7 1 1 1.5 0.5 9.815 11 8 0 1 1.0 1.0 10.55 14 9 1 1 0.5 1.5 10.47 9 10 0 1 1.0 1.0 10.64 19 11 -1 1 1.0 1.5 10.09 25 12 1 1 0.5 1.5 10.1 23 13 1 1 0.5 0.5 10.39 29 14 -1 1 1.0 0.5 10.54 26 15 1 1 1.5 1.5 9.848 8 16 -1 1 1.0 1.5 10.25 16 17 -1 1 0.5 1.0 10.34 12 18 1 1 0.5 0.5 10.27 27 19 -1 1 0.5 1.0 9.663 33 20 0 1 1.0 1.0 10.39 17 21 -1 1 1.5 1.0 9.571 18 22 -1 1 1.0 0.5 10.44 3 23 1 1 0.5 1.5 9.781 15 24 1 1 1.5 1.5 9.848 20 25 0 1 1.0 1.0 10.57 10 26 0 1 1.0 1.0 10.58 5 27 -1 1 0.5 1.0 10.47 32 28 0 1 1.0 1.0 10.52 7 29 -1 1 1.0 0.5 10.52 31 30 0 1 1.0 1.0 10.47 21 31 0 1 1.0 1.0 10.49 22 32 0 1 1.0 1.0 10.39 1 33 1 1 0.5 0.5 10.52 Plant growth hormones used as are 6-benzylaminopurine (BAP) and β-indole acetic acid (IAA).
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Table 1.4 The experimental data of verification: Growth rate of F. velutipes grown on MEA supplementes with concentration of BAP (0.5mg/L) and IAA (0.5mg/L) Diameter (mm) Growth rate Day 3 4 5 6 7 8 (mm/day) 1 34 43 56 68 75 82 10.76 2 34 43 54 65 74 81 10.55 Replicate 31 42 52 63 72 80 10.26 no. 3 4 32 44 56 68 75 84 10.83 5 30 39 51 63 72 82 10.24 Average 10.53 Standard deviation 0.27
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2.0 Selection of Various Lignocellulosic By-products Residues as The Base Carbon- source for Fruiting Substrate of F. velutipes
Table 2.1 The experimental data of the effect of base carbon-source substrates on the radial mycelia growth rate of F. velutipes (mm/day). Ratio Mycelial growth rate (mm/day) mixtures C:N Substrates Hg by weight ratio Replicate Average Stdev (%) 4.76 SD 100 94.72 5.18 5.11 0.32 a 5.39 4.65 PS 100 110.57 4.50 4.60 0.09 b 4.65 6.31 EFB 100 249.19 5.74 6.17 0.39 cd 6.47 6.57 PPF 100 140.42 7.06 6.63 0.40 d 6.27 6.66 75:25 95.34 6.66 6.70 0.06 d 6.77 6.33 SD+PS 50:50 101.66 6.70 6.78 0.49 de 7.31 5.80 25:75 105.81 5.82 5.88 0.12 cd 6.01 6.78 75:25 115.16 6.60 6.70 0.09 de 6.70 6.69 SD+EFB 50:50 138.87 6.77 6.72 0.04 de 6.70 6.41 25:75 177.18 6.26 6.35 0.08 d 6.38 7.22 75:25 102.41 7.20 7.20 0.02 e 7.18 SD+PPF 6.91 50:50 113.00 6.86 6.88 0.03 de 6.88
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6.32 25:75 123.90 6.21 6.27 0.06 d 6.28 4.76 75:25 129.81 5.17 5.06 0.26 a 5.26 5.78 PS+EFB 50:50 157.66 4.26 5.06 0.76 a 5.13 6.18 25:75 192.51 6.18 6.13 0.09 cd 6.02 6.41 75:25 116.34 6.32 6.33 0.07 d 6.27 6.47 PS+PPF 50:50 124.35 7.06 6.84 0.32 de 6.99 6.43 25:75 131.02 6.51 6.47 0.04 d 6.47 6.58 75:25 210.36 6.36 6.46 0.11 cd 6.43 6.34 EFB+PPF 50:50 181.21 6.37 6.26 0.16 cd 6.08 6.65 25:75 158.56 6.78 6.64 0.14 de 6.50 StDev is standard deviation. The same letters denotes insignificant statistical differences (P≤0.05). Materials used as are sawdust (SD), paddy straw (PS), empty fruit bunches (EFB), palm pressed fiber (PPF).
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Table 2.2 The experimental data of the effect of selected base carbon-source substrates on the mycelia growth rate (mm/day) and mycelia thickness. Time for Wet Substrate pH Mycelial growth rate (mm/day) complete Mycelial substrate C:N (%) spawn thickness weight (g) Replicate Mean StDev Hg Replicate Mean StDev Hg run SD + EFB a) 465.28 6.29 1.84 49 (50:50) b) 439.89 138.87 6.37 6.33 0.04 a 1.61 1.76 0.13 b 49 dense c) 473.68 6.34 1.83 49 SD + PS a) 337.69 6.74 1.96 44 (50:50) b) 323.25 101.66 6.80 6.76 0.03 b 2.18 2.05 0.11 bc 44 sparse c) 337.07 6.74 2.02 45
PS + EFB a) 341.18 6.40 1.19 50 (25:75) b) 375.92 192.51 6.41 6.42 0.03 c 1.33 1.26 0.07 a 50 dense c) 333.00 6.45 1.24 50 SD + PPF a) 436.74 5.51 2.59 31 (75:25) b) 445.52 102.41 5.56 5.54 0.03 d 1.59 1.90 0.6 b 45 sparse c) 405.40 5.54 1.51 45
PS + PPF a) 423.10 5.28 1.07 50 (50:50) b)364.50 124.35 5.32 5.33 0.06 e 1.40 1.25 0.17 a 50 sparse c) 398.74 5.39 1.27 50 EFB + PPF a) 546.50 5.05 1.56 49 (25:75) b) 560.33 158.56 5.09 5.06 0.02 f 1.71 1.63 0.07 ab 49 dense c) 550.42 5.05 1.62 49
PPF (100) a)636.48 4.71 1.72 49 b) 695.47 140.42 4.72 4.71 0.01 g 1.85 1.79 0.07 ab 49 dense
c) 671.74 4.71 1.80 49 StDev is standard deviation. Materials used as are sawdust (SD), paddy straw (PS), empty fruit bunches (EFB), palm press fiber (PPF). The same letters denotes insignificant statistical differences (P≤0.05).
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Table 2.3 The experimental data of the effect of selected base carbon-source substrates on the yield of basidiocarps (g) and Biological Efficiency, BE (%). Wet Dry Basidiocarp yield (g) Substrate B.E. (%) substrate C:N substrate 1st 2nd Total (%) weight (g) weight (g) flush flush Replicate Mean StDev Hg Replicate Mean Stdev Hg SD + EFB a) 465.28 46.53 63.17 13.58 76.75 164.95 (50:50) b) 439.89 138.87 43.99 30.68 6.97 37.65 57.91 19.59 a 85.59 125.27 39.68 a c) 473.68 47.37 49 10.34 59.34 125.27 SD + PS a) 337.69 33.77 33.5 9.92 43.42 128.58 (50:50) b) 323.25 101.66 32.32 31.44 9.73 41.17 41.21 2.20 ab 127.38 123.91 7.07 a c) 337.07 33.71 30.1 8.93 39.03 115.78
PS + EFB a) 341.18 34.12 55.89 16.38 72.27 211.81 (25:75) b) 375.92 192.51 37.59 59.56 15.84 75.40 65.08 15.24 ac 200.59 185.09 36.98 ab c) 333.00 33.3 36.59 10.99 47.58 142.88 SD + PPF a) 436.74 43.67 28.57 6.54 35.11 80.40 (75:25) b) 445.52 102.41 44.59 28.96 6.5 35.46 32.08 5.55 ab 79.52 74.41 9.62 ac c) 405.40 40.54 20.59 5.08 25.67 63.32
PS + PPF a) 423.10 42.31 27.8 12.12 39.92 94.35 (50:50) b)364.50 124.35 36.45 47.9 13.14 61.04 59.02 18.17 a 167.46 150.89 50.35 ab c) 398.74 39.87 60.84 15.26 76.10 190.87 EFB + PPF a) 546.50 54.65 54.72 10.01 64.73 118.44 (25:75) b) 560.33 158.56 56.03 50.18 8.96 59.14 41.29 35.87 ab 105.55 74.67 64.98 ac c) 550.42 55.04 0 0 0.00 0.00
PPF (100) a)636.48 63.65 77.42 12.16 89.58 140.74 b) 695.47 140.42 69.55 68.6 9.86 78.46 85.93 6.47 c 112.81 129.06 14.51 c
c) 671.74 67.17 78.13 11.63 89.76 133.63 StDev is standard deviation. Materials used as are sawdust (SD), paddy straw (PS), empty fruit bunches (EFB), palm press fiber (PPF). The same letters denotes insignificant statistical differences (P≤0.05).
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3.0 Effect of Supplementation of Nitrogen-source By-product on Mycelial Growth and Yield of Flammulina velutipes
Table 3.1 The experimental data of the effect of SD+RB (80:20) on mycelia growth rate (mm/day), yield of F. velutipes basidiocarps (g) and Biological Efficiency, B.E. (%). Mycelia Yield (g) Wet Dry Replicate growth B.E. substrate substrate no. rate (%) weight (g) weight (g) 1st flush 2nd flush Total (mm/day) 1 568.77 56.88 1.88 41.88 14.53 56.40 99.16 2 523.48 52.35 1.85 31.01 0.00 31.01 59.24 3 432.10 43.21 1.65 26.89 5.09 31.98 74.00
Table 3.2 The experimental data of the effect of PS+EFB (25:75) on mycelia growth rate (mm/day), yield of F. velutipes basidiocarps (g) and Biological Efficiency, B.E. (%). Supplements Wet Dry Mycelia Yield (g) (%) substrate substrate growth B.E. weight weight rate (%) RB SY 1st flush 2nd flush Total (g) (g) (mm/day) 426.11 42.61 1.96 47.61 n/a 47.61 111.74 20.0 20.0 439.21 43.92 2.01 14.19 18.84 33.03 75.2 482.93 48.29 2.02 43.49 n/a 43.49 90.04 417.75 41.78 2.59 25.84 12.59 38.43 91.99 5.0 5.0 416.86 41.69 2.32 22.12 n/a 22.12 53.06 434.60 43.46 2.26 22.89 n/a 22.89 52.67 405.77 40.58 1.98 69.48 n/a 69.48 171.23 12.5 12.5 410.17 41.02 2.14 35.05 3.17 38.22 93.17 397.70 39.77 2.06 55.66 n/a 55.66 139.95 580.33 58.03 1.57 22.89 71.76 94.65 163.1 20.0 5.0 601.85 60.19 2.04 50.45 7.26 57.71 95.89 535.30 53.53 1.99 20.6 48.68 69.28 129.42 438.00 43.80 2.16 14.01 n/a 14.01 31.99 5.0 20.0 448.18 44.82 2.07 19.75 n/a 19.75 44.07 448.40 44.84 2.13 48.53 n/a 48.53 108.23 Materials used as are rice bran (RB) and spent yeast (SY).n/a: not available
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Table 3.3 The experimental data of the effect of PS+PPF (50:50) on mycelia growth rate (mm/day), yield of F. velutipes basidiocarps (g) and Biological Efficiency, B.E. (%). Supplements (%) Wet Dry Mycelia Yield (g) substrate substrate growth B.E. RB SY weight weight rate 1st flush 2nd flush Total (%) (g) (g) (mm/day) 550.80 55.08 1.73 57.02 n/a 57.02 103.52 20.0 20.0 603.70 60.37 1.67 84.6 n/a 84.6 140.13 588.76 58.88 1.68 68.89 22.4 91.28 155.04 438.01 43.8 1.67 59.02 15.46 74.48 170.04 5.0 5.0 533.75 53.38 1.76 79.39 n/a 79.39 148.74 523.44 52.34 1.49 42.7 22.12 64.82 123.83 403.45 40.35 1.77 71.86 17.18 89.04 220.7 12.5 12.5 378.69 37.87 1.92 44.86 6.71 51.56 136.16 590.79 59.08 1.48 34.38 n/a 34.38 58.19 513.41 51.34 2.11 21.69 n/a 21.69 42.25 20.0 5.0 493.74 49.37 2.16 16.4 7.27 23.67 47.94 493.4 49.34 2.11 9.44 17.26 26.7 54.11 444.70 44.47 1.67 59.01 7.9 66.91 150.46 5.0 20.0 587.95 58.8 1.77 66.8 24.12 90.92 154.64 602.54 60.25 1.56 64.65 n/a 64.65 107.3 Materials used as are rice bran (RB) and spent yeast (SY).n/a: not available
Table 3.4 The experimental data of the effect of PPF (100) on mycelia growth rate (mm/day), yield of F. velutipes basidiocarps (g) and Biological Efficiency, B.E. (%). Supplements Wet Dry Mycelia Yield (g) (%) substrat substrat growth B.E. e weight e weight rate 1st 2nd (%) RB SY Total (g) (g) (mm/day) flush flush 749.17 74.92 2.75 27.45 5.84 33.29 44.44 20.0 20.0 724.24 72.42 1.84 65.73 n/a 65.73 90.76 725.38 72.54 1.93 61.46 5.59 67.05 92.44 595.27 59.53 2.00 41.16 5.09 46.25 77.7 5.0 5.0 632.09 63.21 2.01 56.89 0.99 57.88 91.57 614.20 61.42 1.95 30.3 4.72 35.02 57.02 643.79 64.38 2.14 55.01 2.53 57.54 89.38 12.5 12.5 620.79 62.08 2.17 47.23 5.42 52.65 84.81 605.56 60.56 2.18 54.64 1.10 55.74 92.04 104.4 676.78 67.68 1.87 63.21 7.5 70.71 8 20.0 5.0 715.10 71.51 2.07 63.45 2.95 66.4 92.85 786.30 78.63 2.12 61.14 9.73 70.87 90.12 667.35 66.74 2.14 35.57 4.54 40.11 60.1 5.0 20.0 651.08 65.11 2.19 21.53 24.12 45.65 70.11 695.74 69.57 2.32 44.06 1.71 45.77 65.79 Materials used as are rice bran (RB) and spent yeast (SY).n/a: not available
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APPENDIX D: STATISTICAL ANALYSIS
1.0 Effect of Plant Growth Hormones on Mycelia Growth of Flammulina velutipes on Malt Extract Agar (MEA)
1.1 The statistical data of one-way ANOVA on screening between mycelia growth rate (mm/day) with different plant growth hormones concentrations (mg/L). One-way ANOVA: Mycelia growth rate (mm/day) versus Concentration (mg/L)
Source DF SS MS F P Concentration 4 0.39863 0.09966 42.35 0.000 Error 10 0.02353 0.00235 Total 14 0.42216
S = 0.04851 R-Sq = 94.43% R-Sq(adj) = 92.20%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ---+------+------+------+------1 3 10.4967 0.0643 (---*---) 2 3 10.3400 0.0265 (---*---) 3 3 10.1933 0.0404 (----*---) 4 3 10.0900 0.0721 (---*---) 5 3 10.0600 0.0100 (---*---) ---+------+------+------+------10.05 10.20 10.35 10.50
Pooled StDev = 0.0485 Level indicates 1: 1.0 mg/L BAP + 1.0 mg/L IAA 4:1.0 mg/L BAP + 10.0 mg/L IAA 2:5.5 mg/L BAP + 5.5 mg/L IAA 5:10.0 mg/L BAP + 1.0 mg/L IAA 3:10.0 mg/L BAP + 10.0 mg/L IAA
1.2 The statistical data of one-way ANOVA on optimization between mycelia growth rate (mm/day) with different plant growth hormones concentrations (mg/L). One-way ANOVA: Mycelia growth rate (mm/day) versus Concentrations (mg/L)
Source DF SS MS F P Concentrations 8 3.0100 0.3763 8.08 0.000 Error 18 0.8378 0.0465 Total 26 3.8478
S = 0.2157 R-Sq = 78.23% R-Sq(adj) = 68.55%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ------+------+------+------+-- 1 3 9.842 0.010 (----*----) 2 3 9.400 0.153 (----*----) 3 3 9.972 0.175 (----*-----) 4 3 10.094 0.155 (----*----) 5 3 10.527 0.127 (-----*----) 6 3 10.500 0.053 (----*----) 7 3 10.117 0.345 (----*-----) 8 3 10.158 0.433 (----*----) 9 3 10.393 0.125 (----*----) ------+------+------+------+-- 9.50 10.00 10.50 11.00
Pooled StDev = 0.216 106
Level indicates 1: 1.5 mg/L BAP + 1.5 mg/L IAA 6:1.0 mg/L BAP + 0.5 mg/L IAA 2:1.5 mg/L BAP + 1.0 mg/L IAA 7:0.5 mg/L BAP + 1.5 mg/L IAA 3:1.5 mg/L BAP + 0.5 mg/L IAA 8:0.5 mg/L BAP + 1.0 mg/L IAA 4:1.0 mg/L BAP + 1.5 mg/L IAA 9:0.5 mg/L BAP + 0.5 mg/L IAA 5:1.0 mg/L BAP + 1.0 mg/L IAA
2.0 Selection of Various Lignocellulosic By-products Residues as The Base Carbon- source for Fruiting Substrate of F. velutipes
2.1 The statistical data of one-way ANOVA between mycelia growth rate (mm/day) with different substrate formulation (%) One-way ANOVA: Mycelia growth rate (mm/day) versus Substrate formulation (%)
Source DF SS MS F P Formulation 21 29.9635 1.4268 20.55 0.000 Error 44 3.0545 0.0694 Total 65 33.0181
S = 0.2635 R-Sq = 90.75% R-Sq(adj) = 86.33%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ------+------+------+------+-- 1 3 6.6347 0.4003 (--*--) 2 3 6.1713 0.3851 (--*--) 3 3 4.5987 0.0898 (--*--) 4 3 5.1083 0.3215 (--*--) 5 3 6.6960 0.0650 (--*--) 6 3 6.7787 0.4950 (--*--) 7 3 5.8750 0.1158 (--*--) 8 3 6.6960 0.0911 (--*--) 9 3 6.7163 0.0434 (--*--) 10 3 6.3497 0.0811 (--*---) 11 3 7.2020 0.0220 (--*--) 12 3 6.8830 0.0255 (--*--) 13 3 6.2677 0.0576 (--*--) 14 3 5.0610 0.2620 (--*--) 15 3 5.0567 0.7611 (--*--) 16 3 6.1307 0.0924 (--*--) 17 3 6.3307 0.0689 (--*--) 18 3 6.8410 0.3211 (--*--) 19 3 6.4717 0.0400 (--*--) 20 3 6.4567 0.1147 (--*--) 21 3 6.2633 0.1575 (--*--) 22 3 6.6427 0.1401 (--*--) ------+------+------+------+-- 5.0 6.0 7.0 8.0
Pooled StDev = 0.2635 Level indicates 1: SD (100) 9: SD+EFB (50:50) 17: PS+PPF (75:25) 2: PS (100) 10: SD+EFB (25:75) 18: PS+PPF (50:50) 3: EFB (100) 11: SD+PPF (75:25) 19: PS+PPF (25:75) 4: PPF (100) 12: SD+PPF (50:50) 20: EFB+PPF (75:25) 5: SD+PS (75:25) 13: SD+PPF (25:75) 21: EFB+PPF (50:50) 6: SD+PS (50:50) 14: PS+EFB (75:25) 22: EFB+PPF (25:75) 7: SD+PS (25:75) 15: PS+EFB (50:50) 8: SD+EFB (75:25) 16: PS+EFB (25:75)
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2.2 The statistical data of one-way ANOVA between pH with selected substrate formulation (%) One-way ANOVA: pH versus Substrate formulation (%)
Source DF SS MS F P Formulation 6 10.72840 1.78807 1597.85 0.000 Error 14 0.01567 0.00112 Total 20 10.74407
S = 0.03345 R-Sq = 99.85% R-Sq(adj) = 99.79%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev --+------+------+------+------1 3 6.3333 0.0404 (* 2 3 6.7600 0.0346 (* 3 3 6.4200 0.0265 (*) 4 3 5.5367 0.0252 *) 5 3 5.3300 0.0557 (*) 6 3 5.0633 0.0231 *) 7 3 4.7133 0.0058 (* --+------+------+------+------4.80 5.40 6.00 6.60
Pooled StDev = 0.0335 Level indicates 1: SD+EFB (50:50) 3: PS+EFB (25:75) 5: PS+PPF (50:50) 7: PPF (100) 2: SD+PS (50:50) 4: SD+PPF (75:25) 6: EFB+PPF (25:75)
2.3 The statistical data of one-way ANOVA between mycelia growth rate (mm/day) with selected substrate formulation (%) One-way ANOVA: Mycelia growth rate (mm/day) versus Substrate formulation (%)
Source DF SS MS F P Formulation 7 2.6141 0.3734 6.39 0.001 Error 16 0.9356 0.0585 Total 23 3.5496
S = 0.2418 R-Sq = 73.64% R-Sq(adj) = 62.11%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ------+------+------+------+--- 1 3 2.2463 0.1809 (------*------) 2 3 1.7613 0.1295 (------*------) 3 3 2.0533 0.1147 (------*------) 4 3 1.2563 0.0737 (------*------) 5 3 1.8967 0.6017 (------*------) 6 3 1.2470 0.1665 (------*------) 7 3 1.6297 0.0748 (------*------) ------+------+------+------+--- 1.20 1.60 2.00 2.40
Pooled StDev = 0.2418 Level indicates 1: SD+EFB (50:50) 3: PS+EFB (25:75) 5: PS+PPF (50:50) 7: PPF (100) 2: SD+PS (50:50) 4: SD+PPF (75:25) 6: EFB+PPF (25:75)
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2.4 The statistical data of one-way ANOVA between basidiocarp yield (g) with selected substrate formulation (%) One-way ANOVA: Basidiocarp yield (g) versus Substrate formulation (%)
Source DF SS MS F P Formulation 6 5958 993 3.01 0.042 Error 14 4621 330 Total 20 10578
S = 18.17 R-Sq = 56.32% R-Sq(adj) = 37.60%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ------+------+------+------+--- 1 3 57.91 19.59 (------*------) 2 3 41.21 2.20 (------*------) 3 3 65.08 15.24 (------*------) 4 3 32.08 5.55 (------*------) 5 3 59.02 18.17 (------*------) 6 3 41.29 35.87 (------*------) 7 3 85.93 6.47 (------*------) ------+------+------+------+--- 25 50 75 100
Pooled StDev = 18.17 Level indicates 1: SD+EFB (50:50) 3: PS+EFB (25:75) 5: PS+PPF (50:50) 7: PPF (100) 2: SD+PS (50:50) 4: SD+PPF (75:25) 6: EFB+PPF (25:75)
2.5 The statistical data of one-way ANOVA between Biological Efficiency, B.E. (%) with selected substrate formulation (%) One-way ANOVA: B.E. (%) versus Substrate formulation (%)
Source DF SS MS F P Formulation 6 28118 4686 3.26 0.032 Error 14 20106 1436 Total 20 48224
S = 37.90 R-Sq = 58.31% R-Sq(adj) = 40.44%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev -----+------+------+------+---- 1 3 125.27 39.68 (------*------) 2 3 123.91 7.07 (------*------) 3 3 185.09 36.99 (------*------) 4 3 74.41 9.62 (------*------) 5 3 150.89 50.35 (------*------) 6 3 74.66 64.98 (------*------) 7 3 129.06 14.51 (------*------) -----+------+------+------+---- 60 120 180 240
Pooled StDev = 37.90 Level indicates 1: SD+EFB (50:50) 3: PS+EFB (25:75) 5: PS+PPF (50:50) 7: PPF (100) 2: SD+PS (50:50) 4: SD+PPF (75:25) 6: EFB+PPF (25:75)
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3.0 Effect of Supplementation of Nitrogen-source By-product on Mycelial Growth and Yield of Flammulina velutipes
3.1 Main carbon-source medium: PS+EFB (25:75)
3.1.1 The statistical data of one-way ANOVA between mycelia growth rate (mm/day) with different percentage of supplementation (%) One-way ANOVA: Mycelia growth rate (mm/day) versus Supplementation (%)
Source DF SS MS F P Supplementation 5 1.88783 0.37757 142.48 0.000 Error 12 0.03180 0.00265 Total 17 1.91963
S = 0.05148 R-Sq = 98.34% R-Sq(adj) = 97.65%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev +------+------+------+------0 3 1.2600 0.0755 (-*-) 1 3 1.9967 0.0321 (--*-) 2 3 2.2833 0.0321 (-*-) 3 3 2.0600 0.0800 (-*-) 4 3 2.0233 0.0289 (-*--) 5 3 2.1000 0.0300 (-*-) +------+------+------+------1.20 1.50 1.80 2.10
Pooled StDev = 0.0515 Level indicates 0: no supplementation 2: RB+SY (5.0:5.0) 4: RB+SY (20.0:5.0) 1: RB+SY (20.0:20.0) 3: RB+SY (12.5:12.5) 5: RB+SY (5.0:20.0)
3.1.2 The statistical data of one-way ANOVA between basidiocarp yield (g) with different percentage of supplementation (%) One-way ANOVA: Yield (g) versus Supplementation (%)
Source DF SS MS F P Supplementation 5 5631 1126 5.13 0.010 Error 12 2636 220 Total 17 8267
S = 14.82 R-Sq = 68.12% R-Sq(adj) = 54.83%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ------+------+------+------+--- 0 3 65.08 15.24 (------*------) 1 3 41.38 7.52 (------*------) 2 3 27.81 9.20 (------*------) 3 3 54.45 15.66 (------*------) 4 3 73.88 18.89 (------*------) 5 3 27.43 18.50 (------*------) ------+------+------+------+--- 25 50 75 100
Pooled StDev = 14.82 Level indicates 0: no supplementation 2: RB+SY (5.0:5.0) 4: RB+SY (20.0:5.0) 1: RB+SY (20.0:20.0) 3: RB+SY (12.5:12.5) 5: RB+SY (5.0:20.0)
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3.1.3 The statistical data of one-way ANOVA between B.E. (%) with different percentage of supplementation (%) One-way ANOVA: B.E. (%) versus Supplementation (%)
Source DF SS MS F P Supplementation 5 33703 6741 6.16 0.005 Error 12 13136 1095 Total 17 46839
S = 33.09 R-Sq = 71.96% R-Sq(adj) = 60.27%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ------+------+------+------+-- 0 3 185.09 36.99 (------*------) 1 3 92.33 18.38 (------*------) 2 3 65.91 22.59 (------*------) 3 3 134.78 39.29 (-----*------) 4 3 129.47 33.61 (------*------) 5 3 61.43 40.98 (------*------) ------+------+------+------+-- 60 120 180 240
Pooled StDev = 33.09 Level indicates 0: no supplementation 2: RB+SY (5.0:5.0) 4: RB+SY (20.0:5.0) 1: RB+SY (20.0:20.0) 3: RB+SY (12.5:12.5) 5: RB+SY (5.0:20.0)
3.2 Main carbon-source medium: PS+PPF (50:50)
3.2.1 The statistical data of one-way ANOVA between mycelia growth rate (mm/day) with different percentage of supplementation (%) One-way ANOVA: Mycelia growth rate (mm/day) versus Supplementation (%)
Source DF SS MS F P Supplementation 5 1.2224 0.2445 16.26 0.000 Error 12 0.1805 0.0150 Total 17 1.4028
S = 0.1226 R-Sq = 87.14% R-Sq(adj) = 81.78%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ------+------+------+------+ 0 3 1.2467 0.1662 (----*---) 1 3 1.6933 0.0321 (---*----) 2 3 1.6600 0.1054 (---*----) 3 3 1.7300 0.2128 (---*----) 4 3 2.1467 0.0321 (---*----) 5 3 1.6967 0.0643 (---*----) ------+------+------+------+ 1.40 1.75 2.10 2.45
Pooled StDev = 0.1226 Level indicates 0: no supplementation 2: RB+SY (5.0:5.0) 4: RB+SY (20.0:5.0) 1: RB+SY (20.0:20.0) 3: RB+SY (12.5:12.5) 5: RB+SY (5.0:20.0)
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3.2.2 The statistical data of one-way ANOVA between basidiocarp yield (g) with different percentage of supplementation (%) One-way ANOVA: Yield (g) versus Supplementation
Source DF SS MS F P Supplementation 5 5910 1182 4.14 0.020 Error 12 3429 286 Total 17 9339
S = 16.91 R-Sq = 63.28% R-Sq(adj) = 47.98%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ------+------+------+------+ 0 3 59.02 18.17 (------*------) 1 3 77.63 18.16 (------*------) 2 3 72.90 7.41 (------*------) 3 3 58.33 27.95 (------*------) 4 3 24.02 2.52 (------*------) 5 3 74.16 14.56 (------*------) ------+------+------+------+ 25 50 75 100
Pooled StDev = 16.91 Level indicates 0: no supplementation 2: RB+SY (5.0:5.0) 4: RB+SY (20.0:5.0) 1: RB+SY (20.0:20.0) 3: RB+SY (12.5:12.5) 5: RB+SY (5.0:20.0)
3.2.3 The statistical data of one-way ANOVA between B.E. (%) with different percentage of supplementation (%) One-way ANOVA: B.E. (%) versus Supplementation
Source DF SS MS F P Supplementation 5 22450 4490 2.43 0.097 Error 12 22201 1850 Total 17 44651
S = 43.01 R-Sq = 50.28% R-Sq(adj) = 29.56%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev -+------+------+------+------0 3 150.89 50.35 (------*------) 1 3 132.90 26.51 (------*------) 2 3 147.54 23.13 (------*------) 3 3 138.35 81.28 (------*------) 4 3 48.10 5.93 (------*------) 5 3 137.47 26.21 (------*------) -+------+------+------+------0 60 120 180
Pooled StDev = 43.01 Level indicates 0: no supplementation 2: RB+SY (5.0:5.0) 4: RB+SY (20.0:5.0) 1: RB+SY (20.0:20.0) 3: RB+SY (12.5:12.5) 5: RB+SY (5.0:20.0)
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3.3 Main carbon-source medium: PPF (100)
3.3.1 The statistical data of one-way ANOVA between mycelia growth rate (mm/day) with different percentage of supplementation (%) One-way ANOVA: Mycelia growth rate (mm/day) versus Supplementation (%)
Source DF SS MS F P Supplementation 5 0.40958 0.08192 8.68 0.001 Error 12 0.11327 0.00944 Total 17 0.52285
S = 0.09715 R-Sq = 78.34% R-Sq(adj) = 69.31%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ------+------+------+------+-- 0 3 1.7900 0.0656 (-----*------) 1 3 2.0000 0.0608 (-----*-----) 2 3 2.0967 0.2043 (-----*-----) 3 3 2.1633 0.0208 (-----*-----) 4 3 2.1033 0.0289 (-----*-----) 5 3 2.2767 0.0751 (-----*-----) ------+------+------+------+-- 1.80 2.00 2.20 2.40
Pooled StDev = 0.0972 Level indicates 0: no supplementation 2: RB+SY (5.0:5.0) 4: RB+SY (20.0:5.0) 1: RB+SY (20.0:20.0) 3: RB+SY (12.5:12.5) 5: RB+SY (5.0:20.0)
3.3.2 The statistical data of one-way ANOVA between basidiocarp yield (g) with different percentage of supplementation (%) One-way ANOVA: Yield versus Supplementation
Source DF SS MS F P Supplementation 5 4424 885 5.73 0.006 Error 12 1853 154 Total 17 6277
S = 12.43 R-Sq = 70.48% R-Sq(adj) = 58.18%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ------+------+------+------+ 0 3 85.93 6.47 (------*------) 1 3 57.58 23.32 (------*------) 2 3 46.71 15.19 (------*------) 3 3 56.08 2.98 (------*------) 4 3 69.96 0.81 (------*------) 5 3 37.38 10.03 (------*------) ------+------+------+------+ 40 60 80 100
Pooled StDev = 12.43 Level indicates 0: no supplementation 2: RB+SY (5.0:5.0) 4: RB+SY (20.0:5.0) 1: RB+SY (20.0:20.0) 3: RB+SY (12.5:12.5) 5: RB+SY (5.0:20.0)
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3.3.3 The statistical data of one-way ANOVA between B.E. (%) with different percentage of supplementation (%) One-way ANOVA: B.E. (%) versus Supplementation (%)
Source DF SS MS F P Supplementation 5 9089 1818 5.56 0.007 Error 12 3927 327 Total 17 13016
S = 18.09 R-Sq = 69.83% R-Sq(adj) = 57.26%
Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ------+------+------+------+ 0 3 129.06 14.51 (------*------) 1 3 78.72 31.77 (------*------) 2 3 75.78 22.73 (------*------) 3 3 90.02 5.55 (------*------) 4 3 95.95 5.22 (------*------) 5 3 55.71 12.98 (------*------) ------+------+------+------+ 60 90 120 150
Pooled StDev = 18.09 Level indicates 0: no supplementation 1: RB+SY (20.0:20.0) 2: RB+SY (5.0:5.0) 3: RB+SY (12.5:12.5) 4: RB+SY (20.0:5.0) 5: RB+SY (5.0:20.0)
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APPENDIX E: PUBLICATIONS
1.0 Conference: Abstract for poster presented at the International Congress of The Malaysian Society For Microbiology 2011, 8-11 December 2011, Penang, Malaysia.
COMPARATIVE CULTIVATION OF Flammulina velutipes (GOLDEN NEEDLE MUSHROOM) ON AGRICULTURAL LIGNOCELLULOSIC WASTES
Nooraishah Harith, Noorlidah Abdullah, and Vikineswary Sabaratnam
Mushroom Research Centre, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur.
Corresponding author’s email: [email protected]
ABSTRACT
Studies were carried out to cultivate the Golden needle mushroom, Flammulina velutipes, on palm oil mill wastes, such as empty fruit bunches (EFB) and palm pressed fiber (PPF), and paddy straw from rice plantation. Mycelial growth, mycelia thickness and biological efficiency were the parameters evaluated from singular and different combination of substrates. EFB (75%) + PS (25%), PS(50%) + PPF (50%), and PPF (100%) were among the highest biological efficiency, 46.42, 44.92, and 32.17 % respectively. But EFB (75%) + PS (25%) shows the lowest growth rates among all the combinations. The highest yield of 85.94g was obtained when cultivated on 100% PPF. In conclusion, various local agricultural lignocellulsic wastes can be used for the cultivation of F. velutipes.
2.0 Paper submitted to Pesquisa Agropecuaria Brasileira (PAB)
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