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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

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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
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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

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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

vi
TABLE OF CONTENTS

ABSTRACT ii

ABSTRAK iv

ACKNOWLEDGEMENTS vi

TABLE OF CONTENTS vii

LIST OF FIGURES x

LIST OF TABLES xii

LIST OF SYMBOLS AND ABBREVIATIONS xiv

CHAPTER 1.0 INTRODUCTION 1

CHAPTER 2.0 LITERATURE REVIEW 4

2.1 Mushroom Cultivation 4

2.1.1 Inoculum (spawn) production 6

2.1.2 Fruiting substrate formulation 10

2.1.3 Mushroom growing 13

2.2 Flammulina velutipes (Curtis) Singer 13

2.2.1 Morphology 14

2.2.2 Nutritional value and medicinal properties of F. velutipes 16

2.2.3 Environmental factors affecting fruiting of F. velutipes 16

2.3 Agricultural Lignocellulosic Wastes in Malaysia 19

2.3.1 Agroresidues derived from rice cultivation 21

2.3.2 Agroresidues derived from palm oil mill 23

2.3.3 Brewery solid waste 24

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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

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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

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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

xi
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
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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).

18
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.

31
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:

y = b0 + b1x1 + b2x2 + b11x12 + b22x22 + 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
2 2
S = 0.04851122 R = 94.43% R (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
Residual
Percent

0.00
50

-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
3
Frequency

Residual

0.00
2

1 -0.05

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 N ame
A BA P
AB B IA A

Term

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 Ty pe
10.35 Corner
Center
Mean of Growth rate (mm/day)

10.30

10.25

10.20

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
2 2
S = 0.2418 R = 65.9% R (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

Residual
0.0
Percent

50

10 -0.5

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

4.5
Frequency

Residual
0.0
3.0

1.5 -0.5

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

IAA
1.00

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 IA A
0.5
1.0 0.5
BA P 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.

46
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.

48
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
2 2
S = 0.146333 R = 67.81% R (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

Residual
Percent
0.0
50

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) (d)
Histogram of the Residuals Residuals Versus the Order of the Data
4 0.2

3
Frequency

Residual
0.0
2

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 N ame
A RB
B SY

A
Term

AB

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
Mean of Growth rate (mm/day)

2.2

2.1

2.0

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
2 2
S = 0.127932 R = 74.91% R (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
Residual
Percent

0.0
50
-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) (d) Residuals Versus the Order of the Data
Histogram of the Residuals
0.2
4.8
0.1
3.6
Frequency

Residual

0.0
2.4
-0.1
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 N ame
A RB
B SY

A
Term

AB

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

Mean of Growth rate (mm/day)


1.85

1.80

1.75

1.70

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.

61
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
2 2
S = 0.236234 R = 18.25% R (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

62
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 Residuals Versus the Fitted Values
(b)
99 0.6

90 0.4

Residual
Percent

0.2
50
0.0
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
Frequency

Residual

0.2
4
0.0
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 N ame
A RB
B SY

B
Term

AB

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

64
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

65
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.

67
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

68
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,

69
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.

70
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

71
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

72
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).

73
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

75
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.

76
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.

78
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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

95
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.

96
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).

97
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).

98
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
3 31 42 52 63 72 80 10.26
no.
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

99
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

100
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).

101
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 pH Mycelial growth rate (mm/day)
Substrate 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).

102
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).

103
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 1st flush 2nd flush Total (%)
weight (g) weight (g)
(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

104
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

105
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)

107
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)

108
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)

109
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)

110
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)

111
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)

112
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)

113
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)

114
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: noorlidah@um.edu.my

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)

115

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