i

Digitally Signed by: Content manager’s Name DN : CN = Webmaster’s name O = University of Nigeria, Nsukka

OU = Innovation Centre

Ugwoke Oluchi C.

FACULTY OF BIOLOGICAL SCIENCES

THE DEPARTMENT OF PLANT SCIENCE & BIOTECHNOLOGY

EFFECTS OF DIFFERENT ORGANIC WASTES ON THE GROWTH,

YIELD, MARKET QUALITY AND PROTEIN CONTENT OF LENTINUS

SQUARROSULUS (MONT.) SINGER, AN EDIBLE NIGERIAN

MUSHROOM.

OSIBE, DANDY AHAMEFULA

PG/M.Sc./11/58638

ii

TITLE PAGE

EFFECTS OF DIFFERENT ORGANIC WASTES ON THE

GROWTH, YIELD, MARKET QUALITY AND PROTEIN

CONTENT OF LENTINUS SQUARROSULUS (MONT.)

SINGER, AN EDIBLE NIGERIAN MUSHROOM.

iii

CERTIFICATION

Osibe, Dandy Ahamefula, a postgraduate student of Department of Plant Science &

Biotechnology, University of Nigeria, Nsukka, and with Registration No:

PG/M.Sc./11/58638, has satisfactorily completed the requirements for the courses and

research work for the degree of Masters of Science (M.Sc.) in the Department of Plant

Science & Biotechnology. The work contained in the report is original and has not been

submitted in part or in full for any other diploma or degree of this or any other University.

………………………………. ……………………………

PROF. C. E. A. OKEZIE DR. NNEKA V. CHIEJINA

(HEAD OF DEPARTMENT) (SUPERVISOR)

DATE…………………… DATE………………………

………………………………………..

EXTERNAL EXAMINAR

DATE…………………………………

iv

DEDICATION

Dedicated to Mrs. Ann Ekemma Akpa, a fine woman in every sense.

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ACKNOWLEDGEMENTS

Glory is to the Lord God Almighty whose will for me is always perfect.

I wish to express my profound gratitude to Dr. Nneka V. Chiejina, my supervisor, a role

model. Remain blessed Amen.

Special thanks to my lecturers Prof. M. O. Nwosu, Dr. O. S. Udengwu, Dr. (Mrs.) N. E. Abu,

Dr. R. C. Njokuocha, Dr. (Mrs.) C. N. Ogbonna, Dr. C. C. Onyeke, Mrs. C. U. Asuzu and

Mr. E. E. Osayi who assisted me in one way or the other in the course of this project. I am

very grateful. Special thanks are also due Miss. Okeke Ogochukwu of the Department of

Zoology and Environmental Biology, University of Nigeria, Nsukka, for her valuable

assistance in data analysis.

I wish to thank the Botanic garden staff, Mr. Amos Ogbu and Mr. Linus Ugwuja for their

willingness to help when the need arose. Nsukka sawmill workers are highly appreciated for

providing sawdust.

To my colleagues, Mrs. C. Onaebi, Mrs. Faustina Ugwuja, Ukeh Jude, Gambari Uthman,

Obidigbo Chidiebere, Uchenna Egedigwe, Orji Njoku, Bartholomew Ude and Uche Okafor,

thank you for your motivations and encouragements. You are the best companions to work

with and I am happy with you.

My family deserves special thanks indeed, especially my mother Mrs. Catherine Osibe, my

siblings, Uchenna, Dandy Jnr., Kate, Akpa and my cousins, Catherine Eze and Nnenna

Nwogo. I appreciate your love, care and encouragements.

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TABLE OF CONTENTS

Page

Title Page ……………………………………………………………………….. i

Certification …………………………………………………………………….. ii

Dedication ………………………………………………………………………. iii

Acknowledgements ……………………………………………………………... iv

Table of Content ………………………………………………………………… v

Abstract …………………………………………………………………………..vi

List of Tables ……………………………………………………………………..vii

List of Figures ……………………………………………………………………viii

List of Plates ………………………………………………………………………ix

CHAPTER ONE: INTRODUCTION

1.1 Background …………………………………………………………………….1

1.2 Problem Statement ……………………………………………………………..3

1.3 Justification …………………………………………………………………….5

1.4 Objectives of the Study ………………………………………………………..6

CHAPTER TWO: LITERATURE REVIEW …………………………………..7

CHAPTER THREE: MATERIALS AND METHODS

3.1. Sources of Materials ……………………………………………………………12

3.2. Spawn Preparation ……………………………………………………………..12

3.3. Substrate Analysis ……………………………………………………………. 12

3.3.1. Lignin ………………………………………………………………. 12

3.3.2. Cellulose ……………………………………………………………. 13

3.4. Substrate Preparation …………………………………………………………. 13

3.5. Substrate Spawning and Incubation ………………………………………….. 15

3.6. Fruit Body Induction and Harvesting …………………………………………15

3.7. Determination of Mushroom Yield and Biological Efficiency ………………15

3.8. Determination of Mushroom Market Quality………………………………….22

3.9. Protein Analysis ………………………………………………………………...22

3.1.0. Experimental Design and Data Analysis …………………………………….23

CHAPTER FOUR: RESULTS ……………………………………………………24

CHAPTER FIVE: DISCUSSION …………………………………………………35

REFERENCES …………………………………………………………………… 43

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ABSTRACT

Four organic wastes; mahogany (Khaya ivorensis) sawdust (MSD), Gmelina aborea sawdust

(GSD), oil palm fruit fibre (OPFF) and oil palm empty fruit bunch (OPEFB) were evaluated

for their effects on growth, yield, quality and protein content of Lentinus squarrosulus

(Mont.) Singer. Plastic bag technology was used with treatments replicated ten times and

arranged using a completely randomized design. The quality of the harvested mushrooms was

evaluated on the basis of four pileus diameter size groups (>7 cm, 5-7 cm, 3-5 cm, <3 cm)

and a deformed group; while their protein analyses were carried out using Kjeldahl’s method.

Results on mushroom growth showed that oil palm fruit fibre (OPFF) took the least time for

full mycelial colonization and the longest time occurred on Gmelina sawdust (GSD).

Analysis of variance showed that there were significant differences (P < 0.05) in the time

required for primordia initiation of mushrooms grown on oil palm empty fruit bunch

(OPEFB), oil palm fruit fibre (OPFF) and Gmelina sawdust (GSD). Results on mushroom

yield revealed that mean fresh weights of harvested mushrooms varied from 4.12 ± 0.16 g on

oil palm empty fruit bunch (OPEFB) to 16.05 ± 0.68 g on mahogany sawdust (MSD). There

were significant differences (P < 0.05) in the biological efficiency of mushrooms grown on

mahogany sawdust (MSD), Gmelina sawdust (GSD) and oil palm empty fruit bunch

(OPEFB). Mahogany sawdust produced the highest quality mushrooms, with 26% in the >7

cm group while Gmelina sawdust (GSD) and oil palm empty fruit bunch (OPEFB) had none

in the same quality group. The percentage protein content of harvested mushrooms ranged

from 13.27% for mushrooms produced from mahogany sawdust (MSD) to 27.42% for those

grown on oil palm fruit fibre (OPFF). The above findings reveal the possibility of

commercial production of high quality L. squarrosulus on oil palm fruit fibre (OPFF) and

mahogany sawdust (MSD) while oil palm fruit fibre (OPFF) is recommended as the best

substrate for spawn production among the various organic wastes used in this study.

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LIST OF TABLES

1. Analysis of the main constituents of the organic wastes before mushroom

cultivation……………………………………………………………………………25

2. Yield performance of Lentinus squarrosulus on the different organic wastes………29

3. Substrate effect on Lentinus squarrosulus market quality evaluated by cap size groups………………………………………………………………………………...33

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LIST OF FIGURES

1. Effect of different wastes on time for full mycelial colonisation of Lentinus squarrosulus……………………………………………………………………….26

2. Substrate effect on time required for primordia initiation of Lentinus

squarrosulus………………………………………………………………………….27

3. Substrate effect on percentage biological efficiency of harvested mushrooms……30

4. Comparison of the percentage number of basidiocarps of harvested Lentinus squarrosulus in the different market quality groups………………………………………………………………………………32

5. Effect of different wastes on the percentage protein content of harvested mushrooms………………………………………………………………………….34

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LIST OF PLATES

1. Grain spawn of Lentinus squarrosulus…………………………………………….. 14

2. Spawn run (vegetative mycelial growth) of Lentinus squarrosulus on the different wastes……………………………………………………………………………… 16

3. Production of Lentinus squarrosulus on oil palm fruit fibre waste (OPFF)…………17

4. Production of Lentinus squarrosulus on mahogany sawdust (MSD)……………… 18

5. Production of Lentinus squarrosulus on oil palm empty fruit bunch waste (OPEFB)…………………………………………………………………………… 19

6. Production of Lentinus squarrosulus on Gmelina arborea sawdust (GSD)………. 20

7. Harvested mushrooms from the different wastes on the laboratory bench…………21

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CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGROUND

Chang and Miles (1992) defined mushroom as a macrofungus with a distinctive fruiting

body, which can be either epigenous (growing on or close to ground) or hypogenous

(growing under the ground) and large enough to be visible to the naked eye and to be picked

up by hand. Thus, mushrooms need not be only basidiomycetes, or aerial or fleshy, or edible.

Mushrooms can be ascomycetes, grow underground, have a non-fleshy texture and need not

be edible (Chang, 2008).

Mushrooms are widespread in nature and since earliest recorded history; humans have

viewed them as a special kind of food, savoring the delicious flavours and acknowledging the

nutritional value of this special group of fungi (Chang and Buswell, 1996). Mushrooms have

long been appreciated for their flavour and texture, and some for medicinal and tonic

attributes. However, recognition that they are nutritionally a very good food and

physiologically an important potential source of biologically active compounds of medicinal

value is much more recent (Chang, 1996). It is now known that mushrooms are rich in high

quality protein, contain a high proportion of unsaturated fatty acids and have a nucleic acid

content low enough to allow daily use as a form of vegetable (Chang, 1996). Moreover,

latter-day application of modern analytical techniques has, in a number of cases, provided a

scientific basis for assigning medicinal value through the identification of various mushroom-

derived compounds including anti-cancer, anti-viral, immunopotentiating,

hypocholesterolaemic and hepatoprotective agents (Liu et al., 1995). For example, the

pharmacological activities of Ganoderma lucidum have been attributed mainly to triterpenes

and polysaccharides produced by the mushroom. Several polysaccharides and protein-bound

polysaccharides with immune-modulating and anti-tumour activities have also been isolated

from a variety of mushrooms (Chang, 1996).

Agricultural production and the agro-food industry furnish large volumes of solid wastes,

residues and by-products, produced either in the primary agro-forestry sector (crop-based) or

by secondary processing industries (processing-based) with the major part being

lignocellulosic biomass (Philippoussis and Diamantopoulou, 2011). Recently, Zhang (2008),

reviewing the global world information about lignocellulose availability estimated the

production of lignocellulosic biomass to be more than 200x109 tonnes per year. The amount

of crop residues produced annually in the world from 27 food crops is estimated at about

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4x109 tonnes (Lal, 2008). The majority of this organic matter poses an environmental

pollution problem.

In nature, mushrooms have not only been a source of food for man and other animals, but

also have played an important role in the cycling of carbon and other elements through the

breakdown of lignocellulosic plant residues and animal dung, which serve as the substrates

for these saprophytic fungi (Chang, 1996). In this way, mushroom species, as agents of decay

help keep the environment from being overwhelmed by the dead organic debris of plants and

animals. Mushroom forming fungi are therefore amongst nature’s most powerful

decomposers, secreting strong extracellular enzymes due to their aggressive growth and

biomass production (Adenipekun, 2009). They have the capability to produce a wide range of

enzymes that can break down complex substrates into simpler soluble substances and absorb

them for their growth and development (Oei, 1991).

Strong consumer demands and threats of depletion of mushrooms have stimulated increased

worldwide production in the past few decades (Chang and Miles, 2004). The increased

demand for mushrooms is due to their unique culinary and medicinal properties (Yan et al.,

2003). Commercial cultivation of mushrooms as a source of food, nutriceutical and medicine

is now a worldwide industry with over 120 countries contributing to a crop which, in 1999

totalled 4.3 million tonnes (Chang and Miles, 1991). Several reports indicate that commercial

production of fresh edible mushrooms is a rapidly growing industrial activity. In 2002, world

production of cultivated mushrooms was estimated to be 12,250 thousand tonnes and was

valued at about US$ 32 billion, whereas mushroom products used mainly for dietary

supplements were assessed to have generated about US$ 11 billion (Chang, 2006).

Mushroom cultivation is an efficient and relatively short biological process of food protein

recovery from lignocellulosic materials (Martinez-Carrera et al., 2000). The cultivation of

edible mushrooms has become an increasingly important practice in modern society due to

the biotechnological process of bioconversion of various residues into edible mushrooms or

in dietary supplements of high nutritional value, enabling a more efficient utilization of waste

materials. Interestingly, the spent compost that remains after harvesting mushrooms may still

be recycled for use as animal feeds and soil conditioner. Earlier studies have demonstrated

that spent compost of both Volvariella and Pleurotus had increased crude protein content

compared with raw straw (Quimio, 2004).

Mushroom production can be a lucrative cottage industry for low-income rural households in

developing countries (Ferchak and Croucher, 1996). The activity is labour intensive and can

provide full or part-time employment. A small mushroom production business can be

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established with low capital investment and with minimal requirements for space and

equipment.

Mushroom production represents an important opportunity for developing countries,

particularly Nigeria, since innovations in cultivation and post-harvest processing make

possible new opportunities (Ferchak and Croucher, 1996).

1.2 Problem Statement

Nigeria by virtue of her population size generates several tonnes of agricultural, industrial,

municipal and domestic wastes that overwhelms the nation’s waste disposal machinery and

pose an environmental pollution problem (Okhuoya et al., 2010). These so called wastes

constitute a negative factor both in the economic evaluation of existing industrial and

agricultural operations and because of the adverse environmental effects resulting from their

disposal. Sadly, much of this waste is either burned, shredded or used as landfill or for

improvement of soil quality, even though these wastes constitute a potentially valuable

resource and can be recycled for the production of edible food for man (Chang, 1996).

Much of the cellulose in nature is bound physicochemically with lignin. Because lignin is

highly resistant, it protects cellulose against attack by most microbes, and it must be degraded

by chemical or biological means before the cellulose can be utilized (Salvagi and Kaulkarnis,

2001). The use of the polysaccharides in the lignocellulosic complex is also limited due to

their high lignin content (Hadar et al., 1992). Zadrazil and Grabbe (1983) reported that about

one-half the total production of plant residues from agriculture and industrial processes

remains unused and burdens the environment. Chang (1989) also noted that all agricultural

production from crop plants generated enormous waste, because little of each crop was

actually used; typically 80-90% of the total biomass of agricultural production is discarded as

waste and this is because only part of the organic matter synthesized through photosynthesis

every year is directly edible in the form of fruits, vegetables and food grains.

The handling and disposal of these lignocellulosic residues are often problematic due to their

chemical structure and decomposition properties (Philippoussis et al., 2001). However,

various problems associated with the practical utilization of these materials have not yet been

solved (Taniguchi et al., 2005). One of the key problems hindering the effective utilization of

these renewable resources as raw materials for the production of food and feeds is the low

susceptibility of lignocellulose to hydrolysis, which is attributable to the crystalline structure

of cellulose fibrils surrounded by hemicellulose and the presence of the lignin seal which

prevents penetration by degrading enzymes (Chahal and Chahal, 1999).

xiv

Although physical and chemical technologies may, in some cases, play important associated

roles for handling these wastes. Biotechnological approaches are essential for the emergence

of practical conversion processes which can be applied to situations in developing countries

throughout the world where large scale capital intensive operations are inappropriate.

Adequate food-intake is one of the fundamental human requirements, but there is no denying

the fact that millions of people especially in the developing countries of Africa, Asia and

Latin America are beset with danger of their very survival due to its non-availability. The

greatest difficulty in feeding humans is to supply a sufficient quantity of the body building

protein (Chang, 2008). In Africa, the gap between the increasing population and supply of

protein is somewhat wide since the traditional sources of protein have not kept pace with

population growth. In view of the general shortage of animal protein in the developing

countries, the need to explore vegetable protein as an alternative source has been duly

recognized.

An FAO/WHO joint expert group on protein requirement reviewed evidence on the effects of

protein malnutrition on the predisposition of adults and children to infection, on reduced

stature and retarded psychomotor developments in adults malnourished in younger years.

They also observed reduced birth weight and difficulty in recovering from surgery trauma

and other pathological states (Chang, 2008).

Foodstuffs of plant origin such as cereals, vegetables, potatoes and cassava constitute an

important dietary source of protein for many segments of the world’s population particularly

where animal protein is not only in short supply but have become costly and beyond the reach

of middle and poor classes. One of the major disadvantages of these types of foodstuffs is

their low protein contents. As the shortage of high quality protein is at its peak in the

developing countries like Nigeria, there is need to supplement these diets with

unconventional sources of protein.

1.3 Justification

Huge quantities of a wide variety of organic wastes are generated annually through the

activities of the agricultural, forest and processing industries. Therefore, there is considerable

pressure nowadays to develop biotechnological processes for the rational treatment and/or

disposal of these vast quantities of waste lignocellulosic materials generated annually

(Buswell et al., 1996). Of the various approaches adopted, one of the most significant in

terms of producing a higher value product from the wastes is the cultivation of edible

mushrooms by solid state fermentation (Chang and Miles, 1991). Reports have shown that

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various lignocellulosic residues from agro-industrial sector, such as oil palm and timber

wastes among others, can provide the mushroom with nutrients required for spawn run and

fructification which under controlled conditions and procedures result in an optimum product

yield (Fung et al., 2005).

Mushrooms are a nutritious food source being rich in protein, vitamins and minerals. Earlier

reports have shown that mushrooms are rich in ascorbic and amino acids, and protein is their

most abundant nutrient (Fasidi and Kadiri, 1990; Aletor, 1995). The food and agricultural

organization (FAO) recognizes mushrooms as food contributing to the protein nutrition of the

countries which depend largely on cereals because of their high protein quantity and quality

(Kuforiji and Fasidi, 2009). They are also known to contain substances that enhance the

immune system, fight infectious diseases, and lower blood pressure and cholesterol levels.

The fact that mushrooms, a novel source of protein offer a promising way of alleviating

protein malnutrition in developing countries in the nearest future cannot be denied.

In addition, Nigeria by virtue of its vantage tropical location is one of the world’s potential

hotspots for various forms of biological resources including mushroom (Akpaja et al., 2003).

Currently, the exploitation of indigenous Nigerian mycoresources is still over-shadowed by

the preponderance of green plants (Okhuoya et al., 2010). Vigorous researches on these

easily over looked forest members might evolve an accidental source of drugs that would

resolve the world’s cancer, AIDS and leukemia problems (Okhuoya et al., 2010).

1.4 Objectives of the Study

• To determine the effects of different local organic wastes on the growth and yield of

Lentinus squarrosulus.

• To evaluate the market quality of Lentinus squarrosulus cultivated on the different

organic wastes.

• To estimate the protein content of the mushrooms grown on different local organic

wastes.

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CHAPTER TWO

LITERATURE REVIEW

Lentinus squarrosulus is a highly prized Nigerian mushroom, which is appreciated for its

meaty taste and texture (Kadiri, 2005). The mushroom is of immense value in traditional

medicine, and it features considerably into Nigerian folklore and mythology (Oso, 1977). It is

commonly known as ‘Tifa’ in some parts of Nigeria (Oso, 1975), ‘Ero atakata’ by the Igbo

speaking people a name derived from its tough, texture (Akpaja et al., 2003) and ‘Hed Khon

Khao’ in Thailand (Petcharat, 1995). Lentinus squarrosulus is very common in the Southern

part of Nigeria and has been highly recommended for commercialization (Akpaja et al.,

2003). Neda and Doi (1998) reported its wide spread presence throughout Equatorial Africa,

South-East Asia, the Pacific Island and Australia.

Nutritional requirement has been shown to be an important consideration in mushroom

cultivation. The effects of various nutrients and media on the growth and development of

some species of the genus Lentinus have been reported by several authors. While evaluating

media, Gbolagade et al. (2006) reported that potato dextrose agar and yellow corn agar

stimulated the best mycelial extension in Lentinus subnudus. Nwanze et al. (2005) showed

that the interaction of spawn grain and culture medium had a significant effect on carpophore

dry weight, stipe and pileus diameters of L. squarrosulus. Being an achlorophyllus organism,

mushrooms rely entirely on organic carbon for energy source. The correlation between source

of carbon and mycelial growth has been reported by various researchers. Kaur and Lakhanpal

(1995) documented dextrose as the most suitable carbon source for mycelial growth of

Lentinula edodes. Nwanze et al. (2005) reported optimum production of fungal biomass in L.

squarrosulus in liquid culture using glucose and butter as carbon and lipid sources

respectively. Similar to carbon, nitrogen is also reported to be an essential element for the

growth and development of the mycelium. Nitrogen is indispensable for building protoplasm

and cell structural elements in mushrooms (Lopez et al., 2004). Kadiri and Fasidi (1994)

reported peptone as the best nitrogen source for L. subnudus. In the same vein, Luo (1993)

reported that organic nitrogen sources such as yeast extract and peptone are the preferred

nitrogen sources for Auricularia auricular.

The cultivation of edible mushrooms has become an increasingly important practice in

modern society due to the biotechnological process of bioconversion of various residues in

edible mushrooms or in dietary supplement of high nutritional value. Mushrooms are grown

on some organic substrates, mostly waste materials from farms, plantations or factories

(Quimio, 2004). However, identification of suitable substrate materials is critical for

xvii

successful mushroom cultivation (Shah et al., 2004). Understanding the impact of substrate

on mushroom productivity and quality is valuable to determine the combination of suitable

substrate composition and fungal strain that bioconvert effectively the agricultural wastes into

nutritional and medicinal food (Philippoussis and Diamantopoulou, 2011). Various types of

substrates have been reported to be used for the production of edible fungi. Ayodele et al.

(2007) reported that the highest fresh weight of L. squarrosulus was observed on

Brachystegia nigerica sawdust (32.10 ± 1.56 g) and the least was on Terminalia sp sawdust

with 5.20 ± 0.31 g. They also observed the highest mycelial density of L. squarrosulus in the

sawdust of Mansonia altissimia. Ambi et al. (2011) in their studies on cultivation of some

Pleurotus species on lignocellulosic materials showed that Gmelina arborea sawdust is the

most suitable substrate for growing Pleurotus ostreatus with the lowest total fresh weight

recorded for Khaya senegalensis. Chiejina and Olufokunbi (2010) found that Pleurotus

tuberregium was unable to produce fruit bodies on oil palm fruit fibre although the highest

fresh weight yield of the mushroom was obtained from a mixture of sawdust and river sand.

Cultivation studies on Psathyrella atroumbonata a Nigerian edible mushroom on different

agro-industrial wastes showed that the maximum mycelial extension was recorded on oil

palm fruit fibres with the highest sporophore yield of 61.63 ± 0.08 g obtained on sawdust of

mahogany (Ayodele and Okhouya, 2007)

The perception of mushrooms as a highly nutritional food stuff is well founded (Buswell and

Chang, 1993). Compositional analysis of the major cultivated varieties has revealed that, on a

dry weight basis, mushrooms normally contain 19-35% protein (Chang and Buswell, 1996).

The protein content of mushrooms is almost equal to that of corn, milk and legumes although

still lower than meat, fish and eggs (Quimio, 2004). As a dietary source of protein,

mushrooms are superior to most fruits and vegetables with the exception of beans and peas

(Quimio, 2004). Additionally, mushroom proteins contain all the essential amino acids and

are especially rich in lysine and leucine which are lacking in most staple cereal foods (Chang

and Buswell, 1996). The low total fat content and high proportion of polyunsaturated fatty

acids (72 to 85%) relative to total fatty acids is considered as significant contributor to the

health value of mushrooms (Chang and Buswell, 1996). Mushrooms also rank quite high in

their vitamin content, which includes significant amounts of vitamin C (Quimio, 2004).

Although devoid of vitamin A, mushrooms make up for that with their high riboflavin,

thiamine and cyanocobalamin (Vit. B12) content, the latter usually found only in animal

products (Quimio, 2004).

xviii

The proximate, amino acid composition and mineral content of L. squarrosulus have been

reported by many researchers. Fasidi and Kadiri (1990) showed that mature L. squarrosulus

fruit bodies are rich in ascorbic acid and amino acids, and protein is their most abundant

nutrient. Nwanze et al. (2006) reported 22.82% crude protein, 7.64% crude fiber, 7.52% ash,

2.76% moisture, 6.29% crude fat and 60.65% soluble carbohydrates in L. squarrosulus.

Sharma et al. (2012) evaluated some wild edible mushrooms for amino acid composition.

The result showed that aspartic acid content ranged from 0.25-0.37% with maximum in L.

squarrosulus (0.37 %) and minimum in L. torulosus (0.25 %). Arginine content of the

mushrooms ranged from 0.21-0.29% while the alanine content ranged from 0.09-0.15%.

Ayodele et al. (2011) investigated the effects of sun, smoke and oven drying methods on the

nutrient contents of four wild edible mushrooms in Nigeria. They found that nutrient contents

were higher in sun drying methods than oven and smoke drying methods.

Mushrooms have been traditionally used in China and Japan for their medicinal and tonic

properties (Chang, 1996). Labarere and Menini (2000) acknowledged that the uses of

mushroom genetic resources are not only of high interest in agronomy, agriculture, human

food and animal feed but also for the discovery, production and development of molecules or

components with high added value in chemical and pharmaceutical industries. Chihara (1992)

reported that hot water extracts of some basidiomycetes, such as Ganoderma applanatus and

Coriolus versicolor markedly inhibited the growth of sarcoma 180. Chang and Buswell

(1996) reported that Volvariella volvacea is traditionally used in China and Japan for

lowering blood pressure and accelerating the healing of wounds. Recent application of

modern analytical techniques has, in a number of cases, provided a scientific basis for these

earlier empirical observations (Chang and Buswell, 1996). β-D-glucans and proteoglycans

have been isolated from various mushrooms and shown to have anti-cancer activity (Smith et

al., 2002). Several species of the Pleurotus genus produce mevinolin or lovastatin, an

inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase that is used in the

treatment of hypercholesterolemia. The addition of 15-20 g dried Pleurotus each day for 1

month in diets reduced hypercholesterolemia in many but not all patients (Bobek et al.,

1998). PSK (trade name Krestin) a protein-bound polysaccharide extracted from the mycelia

of C. versicolor has been reported to display various unique biological activities including the

stimulation of functional maturation of macrophages, inhibition of the cytopathic effect of

HIV infection and an ability to scavenge active oxygen species (Buswell et al., 1996). Chang

and Buswell (1996) reported that Ganoderma lucidum is able to concentrate the element

xix

germanium at a high concentration equivalent to those found in crude drugs obtained from

ginseng.

One of the major environmental problems facing the world today is the contamination of soil,

water and air by toxic chemicals as a result of industrialization and extensive use of

pesticides in agriculture (Adenipekun and Lawal, 2012). During the vegetative phase of

mushroom life cycle, mycelium grows through the substrate, biodegrades its components and

supports the formation of fruiting bodies (Philippoussis and Diamantopoulou, 2011).

Alexander (1994) reported that the ability of fungi to transform a wide variety of hazardous

chemicals has aroused interest in using them in bioremediation. Fungi are among nature’s

most powerful decomposers, secreting strong enzymes (Adenipekun and Lawal, 2012). L.

squarrosulus has been found to mineralize soil contaminated with various concentrations of

crude oil resulting in increased nutrient contents in treated soil (Adenipekun and Lawal,

2012). Adenipekun and Fasidi (2005) reported the ability of L. squarrosulus to mineralize

soil contaminated with various concentrations of crude oil (1-40%). They found that nutrient

contents were generally higher after 6 months of incubation except potassium levels which

did not increase. The rapid mycelial growth and enhanced enzyme production by L.

squarrosulus have biotechnological applications in wood and pulp, textile, and tanning, as

well as in the bioremediation of oil spills (Adenipekun and Lawal, 2012).

Mushroom in recent times has become a contemporary business enterprise because of its high

nutritional and medicinal values, hence high societal demand (Onuoha et al., 2009). The

mushroom market has grown rapidly in recent years. In the United States, fresh mushroom

production more than quadrupled in 15years, from 1975-1990; total annual production was

743 million pounds in 1991-92; 68% was for the fresh market and 32% was processed

(Ferchak and Croucher, 1996). The world market for the mushroom industry in 2001 was

valued at over US$ 40 billion (Chang, 2006). Production of mushrooms worldwide has been

steadily increasing, mainly due to contributions from developing countries such as China,

India, Poland, Hungary and Vietnam (Chang, 2008). There are also increasing experimentally

based evidence to support centuries of observations regarding the nutritional and medicinal

benefits of mushrooms (Chang, 2008).

Globalization is affecting food production and consumption chains worldwide. Consumers

from many regions are increasingly getting a wider variety of food products of higher quality

and lower price (Mayett et al., 2006). Mushrooms are an important commodity worldwide,

whose production has been increasing steadily. However, a thorough understanding of

consumption trends is not yet available (Mayett et al., 2006). Major efforts in mushroom

xx

cultivation have been focused on technological developments and yields. Therefore, in a

global economy, the future of mushroom cultivation will also depend on a thorough

understanding of the consumption trends worldwide (Mayett et al., 2006). This is particularly

the case in developing countries where the importance of edible mushrooms within consumer

preferences and perceptions is not yet clear and studied (Mayett et al., 2004) and the

mushroom industry is in its infancy (Isikhuemhem and Okhuoya, 1995).

xxi

CHAPTER THREE

MATERIALS AND METHODS

3.1. Sources of Materials

Stock culture of Lentinus squarrosulus used for the experiment was obtained in January 2013

from the Pathology Unit of Forestry Research Institute Ibadan, Nigeria. Fresh hardwood

sawdust of mahogany (Khaya ivorensis) and Gmelina aborea were collected from a local

sawmill in Nsukka, Nigeria while the oil palm empty fruit bunch (OPEFB) and oil palm fruit

fibre (OPFF) were obtained from a local oil palm industry in Nru, Nsukka, Nigeria in

February 2013.

3.2. Spawn Preparation

The spawn was prepared using Sorghum (Sorghum bicolor) grains as substrate. A modified

method of Oghenekaro et al. (2009) was adopted for spawn preparation. Empty salad cream

jars were filled with parboiled sorghum grains (75% water) to three quarter full and covered

with cotton wool. The jars were autoclaved at a temperature of 121 0C and 103 KNM-2

pressure for 30 min and allowed to cool down overnight after which they were inoculated

with the pure culture of Lentinus squarrosulus under aseptic conditions in the inoculation

chamber. The inoculated grains were incubated at room temperature (25 ± 2 0C). After the

grains had been fully colonized (Plate 1), they were stored in a refrigerator at 12 0C until

required.

3.3. Substrate Analysis

The cellulose, hemicellulose and lignin contents of the organic wastes were determined

before mushroom growth by the method of Datta (1981) while the nitrogen content of the

wastes was determined by the method of AOAC (2002).

3.3.1. Lignin: The wastes were extracted with ethanol:benzene 1:2 (v/v) for 4 h, washed with

ethanol and ether and then dried at 45 0C. The residue was refluxed with 150 ml 5% H2SO4

for 1 h. Final washing was carried out with 20 ml hot water and refluxed in 150 ml 3% H2SO4

for 2 h. The amount of lignin was calculated on an ash-free basis.

3.3.2. Cellulose: Chlorine gas was passed into a 2 g moistened waste sample (W1) in a

conical flask, followed (3 times at 5 min interval) by 5 ml 17.5% NaOH solution. Distilled

water (33 ml) was added and sample was filtered, dried at 105 0C for 2 h and weighed (W2).

Fifty milliliter (50 ml) of 8.3% NaOH solution was added and the contents filtered again and

washed four times with distilled water after which it was again filtered and the residue dried

at 105 0C for 2 h. The weight of the residue was calculated as W3.

xxii

Percentage cellulose =

Where W3 = dried weight of the cellulose sample

Percentage holocellulose =

Where W1 = Initial dry weight of the sample and W2 = dried weight of holocellulose in the

sample. The hemicellulose was obtained as the difference between the holocellulose and

cellulose contents.

3.4. Substrate Preparation

A modified method of Chiejina and Olufokunbi (2010) was adopted for substrate preparation.

The fresh OPEFB (chopped into small pieces of about 1-5 cm) and OPFF were soaked in

distilled water overnight in order to wash out the remaining oil in the fibre and to gain 75%

moisture content. The moisture content of the sawdust was adjusted to 75% with distilled

water by sprinkling. Three hundred grams oven-dry-weight equivalent of the moistened

substrates were each filled into ten (10) high porosity polypropylene plastic bags measuring

17.5x15 cm each. A polyvinyl chloride pipe measuring 5 cm wide and 3 cm long was passed

through the top of each bag. Thereafter, the mouth of each bag was plugged with cotton wool

and covered with foil paper. The bags were autoclaved at a temperature of 121 0C and 103

KNM-2 pressure for 30 min.

xxiii

Plate 1: Grain spawn of Lentinus squarrosulus

3.5. Substrate Spawning and Incubation

Autoclaved bags were allowed to cool down to ambient temperature. The bags were

randomly picked and spawned with 25 g spawn per 500 g substrate (5%, w/w) (Quimio et al.,

1990) under aseptic conditions and incubated at a temperature of 28-30 0C (Plate 2).

3.6. Fruit Body Induction and Harvesting

Bags were transferred to a growing room once primordia had formed. Fruit body induction

was achieved by reducing the temperature of the environment by spraying water into the

room and opening of the mouth of the bags. Fruiting bags were sprayed daily with sterile

water using a hand sprinkler and water was placed in a reservoir on the floor to maintain

humidity at about 85%-90%. Fresh air was circulated in the growing room using an electric

fan. The room was lit on a 12 h on/off cycle using an electronic fluorescent lamp of 85 watts.

Fruit bodies were harvested three days after primordia emergence when the lamellae were

fully exposed (Plates 3-6).

xxiv

3.7. Determination of Mushroom Yield and Biological Efficiency

Data were collected on the average number of days for complete mycelial colonization of

substrates, time required for mushroom primordia initiation, fruit body yields (stipe height,

pileus diameter and fresh weight) and biological efficiency (BE). BE was determined as the

ratio of fresh mushrooms harvested (g) per g dry substrate and expressed as a percentage

(Mamiro and Royse, 2008).

Fresh weight of mushroom X 100

Dry weight of substrates

Harvested mushrooms were placed in a paper bag and oven dried at 60 0C for 48 h. Dried

mushrooms were placed on a laboratory bench for 2 h to cool down before weighing (Plate

7). Mushroom weights were determined using an Ohaus weighing balance (Model AR3130,

0.001 g accuracy, Made in England).

Plate 2: Spawn run (vegetative mycelial growth) of Lentinus squarrosulus on the different

wastes

xxv

Plate 3: Production of Lentinus squarrosulus on oil palm fruit fibre waste (OPFF)

OIL PALM FRUIT FIBRE

xxvi

MAHOGANY SAWDUST

SUBSTRATE

xxvii

Plate 4: Production of Lentinus squarrosulus on mahogany sawdust (MSD)

Plate 5: Production of Lentinus squarrosulus on oil palm empty fruit bunch waste (OPEFB)

OIL PALM EMPTY FRUIT

BUNCH

xxviii

Plate 6: Production of Lentinus squarrosulus on Gmelina arborea sawdust (GSD)

GMELINA ARBOREA

SAWDUST

xxix

Plate 7: Harvested mushrooms from the different wastes on the laboratory bench

KEY: GSD = Gmelina sawdust MSD = Mahogany sawdust OPFF = Oil palm fruit fibre OPEFB = Oil palm empty fruit bunch

3.8. DETERMINATION OF MUSHROOM MARKET QUALITY

The quality of the basidiocarps was evaluated on the basis of four (4) pileus diameter size

groups (larger than 7 cm, 5-7 cm, 3-5 cm, less than 3 cm) and a deformed group. Marketable

mushrooms were those that measured more than 3 cm in diameter and mushrooms of highest

commercial value were those that measured more than 5 cm in diameter (Rossi et al., 2003).

3.9. PROTEIN ANALYSIS

The protein analysis was conducted at the National Centre for Energy Research and

Development, University of Nigeria, Nsukka. The mushrooms harvested from the different

wastes were analyzed for their protein content using Micro-Kjeldahl’s method as described in

AOAC (2002). Oven dried mushroom samples of 0.5 g from each waste was weighed into the

Mushroom

s

harvested

from MSD

Mushrooms

harvested

from GSD

Mushroom

s harvested

from OPFF

Mushrooms

harvested

from

OPEFB

xxx

Kjeldahl digestion flask (Model FK500/31, Made in Barloworld UK). One gram (1 g) of the

catalyst mixture (Mixture of 20 g potassium sulphate, 1 g copper sulphate and 0.1 g selenium

powder) was weighed and added into the flask. Fifteen milliliter (15 ml) of Conc. H2SO4 was

also added. The mixture was cautiously heated on a digestion rack in a fume cupboard until a

bluish green clear solution appeared. The digest was allowed to cool and solidify for 24 h

when the colour changed to white. Twenty milliliter (20 ml) of distilled water was added to

the solidified sample to avoid caking. The digest was transferred after several washings into a

100 ml volumetric flask and made up to the mark with distilled water. A 10 ml aliquot was

collected from the digest and put into the Erlenmayer’s flask in the Micro-Kjeldahl

distillation unit. A 100 ml receiver flask containing 5 ml boric acid indicator solution was

placed under the condenser of the distillation apparatus so that the tip was 2 cm inside the

indicator. Sixty milliliter (60 ml) of 40% NaOH was added to the digested sample through a

funnel stop cork. The distillation commenced by closing the steam jet arm of the distillation

apparatus. The distillate was collected in the receiver flask and was titrated with 0.01 M

standard HCL until a pink colour emerged.

Calculation

% Nitrogen = T × M × 0.014 × D.F × 100

G

Where,

T = Titre value

M = Molarity of standard HCL

D.F. = Dilution factor

100 = conversion to %

0.014 = a constant which means that 0.014 is liberated by 1 ml. of 0.01 HCL

G = Weight of sample used

Percentage Protein = % Nitrogen × 6.25

Where,

6.25 = protein constant according to Kjeldahl’s method.

3.1.0. EXPERIMENTAL DESIGN AND DATA ANALYSIS

xxxi

The experiment comprising four substrate types (MSD, GSD, OPFF and OPEFB) was laid

out using a Completely Randomized Design (CRD) with ten replicates per treatment.

Data on mushroom market quality was analyzed using GENSTAT Statistical Package version

3.0. Two way analysis of variance (ANOVA) was used to test for significance while Fisher’s

Least Significant Difference (FSLD) was used to compare means of groups. Results of

mushroom yield and biological efficiency were analyzed by one way ANOVA using SPSS

version 17.0. Mean separation was done using Duncan Multiple Range Test (DMRT). All

analysis was done at 5% level of significance.

xxxii

CHAPTER FOUR

RESULTS

Results of the chemical analyses of the main constituents of the wastes revealed that

mahogany sawdust (MSD) contained the highest percentage cellulose (85.7%) while Gmelina

sawdust had the least percentage cellulose (57.1%) (Table 1). The highest percentage

nitrogen content (1.36%) was obtained from oil palm fruit fibre (OPFF) while Gmelina

sawdust contained the least percentage nitrogen (0.30%) (Table 1). The result of the

experiment on mushroom growth showed that the mean time required for full mycelial

colonization of the organic wastes by the mushroom mycelia varied from 6.40 ± 0.34 days to

15.40 ± 0.54 days. The fastest substrate colonization of 6.40 ± 0.34 days was observed on oil

palm fruit fibre (OPFF) while the slowest 15.40 ± 0.54 days came from Gmelina sawdust

(GSD) (Fig. 1). Analysis of variance indicated that the time required for full mycelial

colonization was significantly different (p < 0.05) in the various organic wastes evaluated

(Fig. 1). The effect of the different organic wastes on the time required for mushroom

primordia initiation is shown in Fig. 2. Pinheads emerged as small rounded lumps that were

grouped at various parts of the substrate surfaces. The wastes had variable effects on the

duration to primordia initiation ranging from 28.20 ± 0.74 to 39.40 ± 0.87 days for Gmelina

sawdust and oil palm fruit fibre respectively. There were no significant differences (p > 0.05)

in the time required for primordia initiation of mushrooms grown on oil palm empty fruit

bunch (OPEFB) and mahogany sawdust (MSD) (Fig. 2).

xxxiii

Table 1. Analysis of the main constituents of the organic wastes before mushroom

cultivation

Substrate Cellulose Hemicellulose Lignin Nitrogen % % % %

OPFF 74.29 12.00 8.50 1.36 OPEFB 72.86 11.50 10.00 0.69 GSD 57.14 8.50 17.50 0.30 MSD 85.71 10.00 19.30 0.49

KEY:

GSD = Gmelina sawdust MSD = Mahogany sawdust OPFF = Oil palm fruit fibre OPEFB = Oil palm empty fruit bunch

Fig. 1: Effect of different wastes on time for full mycelial colonization of L. squarrosulus

xxxiv

Fig. 2: Substrate effect on the time required for primordia initiation of L. squarrosulus

Results on mushroom yield showed that oil palm fruit fibre produced mushrooms with the

highest mean stipe height (4.50 ± 0.15 cm) while the least (2.10 ± 0.07 cm) was from oil

palm empty fruit bunch (Table 2). There were significant differences (p < 0.05) in the mean

stipe height of mushrooms produced from the various organic wastes. Fruit bodies with the

xxxv

widest mean pileus diameter (5.76 ± 0.21 cm) were those grown on mahogany sawdust while

the least (3.01 ± 0.03 cm) was from those grown on oil palm empty fruit bunch (Table 2).

Analysis of variance indicated that there were significant differences (p < 0.05) in the mean

pileus diameter of mushrooms produced from the various wastes evaluated. The effect of the

different organic wastes on mushroom fresh and dry weights is also shown in Table 2.

Mushrooms with the highest mean fresh weight (16.05 ± 0.68 g) were those grown on

mahogany sawdust while oil palm empty fruit bunch produced mushrooms with the least

(4.12 ± 0.16 g) mean fresh weight. There were no significant differences (p > 0.05) in the

mean fresh weight of mushrooms grown on mahogany sawdust and oil palm fruit fibre (Table

2). The highest mean dry weight (2.78 ± 0.22 g) mushrooms were produced from mahogany

sawdust and the least (1.06 ± 0.01 g) from those grown on oil palm empty fruit bunch (Table

2). Results showed that the mean dry weight of mushrooms produced from mahogany

sawdust was significantly higher (p < 0.05) than those of other treatments.

Biological efficiency (BE) was calculated to determine how the mushroom utilized nutrients

present in the various organic wastes evaluated. Fig. 3 shows that mahogany sawdust

produced mushrooms with the highest mean percentage biological efficiency (5.35 ± 0.23 %)

while oil palm empty fruit bunch gave the least (1.37 ± 0.05 %). There were no significant

differences (p > 0.05) in the mean percentage biological efficiency of mushrooms grown on

mahogany sawdust and oil palm fruit fibre.

Table 2. Yield performance of Lentinus squarrosulus on the different organic wastes

Substrate Stipe height

(cm)

Pileus diameter

(cm)

Fresh weight

(g)

Dry weight (g)

MSD 3.17 ± 0.11b 5.76 ± 0.21a 16.05 ± 0.68a 2.78 ± 0.22a

xxxvi

Each value is a mean of 10 replicates. Values in the same column followed by the same letter (s) are not significantly different at p > 0.05 according to Duncan multiple range test (DMRT). KEY:

MSD = Mahogany sawdust GSD = Gmelina sawdust OPFF = Oil palm fruit fibre OPEFB = Oil palm empty fruit bunch

Fig. 3: Substrate effect on percentage biological efficiency of harvested mushrooms

GSD 2.46 ± 0.05c 4.06 ± 0.10c 9.02 ± 0.33b 1.56 ± 0.08b

OPFF

4.50 ± 0.15a

4.86 ± 0.22b

15.10 ± 0.72a

1.31 ± 0.08bc

OPEFB

2.10 ± 0.07d

3.01 ± 0.03d

4.12 ± 0.16c

1.06 ± 0.01c

xxxvii

Mushroom market quality in terms of basidiocarp pileus diameter was affected by substrate

type. The market quality size group 3-5 cm, had the highest mean percentage number of

basidiocarp (56.50%) in all wastes evaluated while the deformed group had the least (0.00%)

percentage number of basidiocarp (Fig. 4). There were significant differences (p < 0.05) in

the mean percentage number of basidiocarps in the market quality size groups >7, 3-5 cm and

the deformed group as shown in Fig. 4. However, there were no significant differences (p >

0.05) in the mean percentage number of basidiocarps in the market quality groups <3 and 5-7

cm. Mahogany sawdust yielded the highest quality mushrooms, with 26% in the >7 cm group

while Gmelina sawdust and oil palm empty fruit bunch had none in the same quality group

(Table 3). Gmelina sawdust stimulated highest production of basidiocarp in the 3-5 cm size

group. Oil palm empty fruit bunch had many of the basidiocarps in the 3-5 and <3 cm size

groups with 56% and 44% respectively (Table 3). It is noteworthy that no fruit bodies were

deformed throughout the experiment and hence none recorded for the deformed group.

The percentage protein content of harvested mushrooms varied significantly as a result of the

effect of the various organic wastes. Fig. 5 shows that the percentage protein content ranged

from 13.27% for mushrooms produced from mahogany sawdust to 27.42% for those grown

on oil palm fruit fibre.

xxxviii

Fig. 4: Comparison of the percentage number of basidiocarps of harvested Lentinus

squarrosulus in the different market quality groups

Table 3. Substrate effect on Lentinus squarrosulus market quality evaluated by cap size

groups

Market quality groups (cm)

Substrate

<3 cm 3-5 cm 5-7 cm >7 cm Deformed

GSD MSD OPEFB OPFF

8.00b2

82.00a1

10.00b2

0.00b2

0.00a2

14.00b2

36.00c1

34.00a1

26.00a1

0.00a3

44.00a2

56.00b1

0.00b3

0.00b3

0.00a3

8.00b3

52.00b1

30.00a2

10.00b3

0.00a3

LSD(5%)

11.370

Values are expressed as a percentage of the pileus diameter and represent the mean of 10 replicates. Mean values in the same row followed by the same figure (s) are not significantly

xxxix

different at p > 0.05. Mean values in the same column followed by the same alphabet (s) are not significantly different at p > 0.05 according to Fischer’s Least Significance Difference. KEY: MSD = Mahogany sawdust GSD = Gmelina sawdust OPFF = Oil palm fruit fibre. OPEFB = Oil palm empty fruit bunch

Fig. 5: Effect of different wastes on the percentage protein content of harvested mushrooms

KEY:

GSD = Gmelina sawdust MSD = Mahogany sawdust OPEFB = Oil palm empty fruit bunch OPFF = Oil palm fruit fibre.

CHAPTER FIVE

DISCUSSION

The cultivation of edible mushrooms serves as the most efficient and economically viable

biotechnology for the conversion of lignocellulosic waste materials into food of high

nutritional value (Fasidi and Ekuere, 1993). One of the strongest technical points recently

advancing mushroom production in Nigeria besides improving food options is the conversion

of valueless or toxic wastes of diverse origin to value added products via a permaculture

system (Okhuoya et al., 2010). The results of our study showed that L. squarrosulus mycelia

xl

grew satisfactorily on all the organic wastes evaluated. This agrees with the findings of

Kimenju et al. (2009) who demonstrated that mushrooms can be grown on several locally

available organic substrates. In the same vein, Fasidi and Kadiri (1993) and Ayodele et al.

(2007) found that Lentinus spp. could be cultivated on a wide variety of agricultural wastes.

Philippoussis et al. (2003) revealed that the type of wastes, as well as the strain of the fungus

used exercised a considerable influence on the colonization rates and therefore on the time

needed for complete colonization and fructification of Lentinula edodes. Mushroom mycelia

are also known to produce significant quantities of a plethora of enzymes, which can degrade

the lignocellulosic residues and use them as nutrients for their growth and fructification

(Elisashvili et al., 2008).

Vigorous substrate colonization by the mycelium during spawn run is desirable because it

reduces mushroom cropping time and may allow mycelium to outgrow competitors in the

substrate (Mamiro and Royse, 2008). In the present study, the least time taken for full

mycelial colonization of the organic wastes was recorded on oil palm fruit fibre (OPFF). This

agrees with the results of Ayodele and Okhuoya (2007) who reported the fastest mycelial

extension of Psathyrella atroumbonata to be on OPFF. Ayodele and Akpaja (2007) obtained

enhanced mycelial growth and sporophore yield of L. squarrosulus on sawdust supplemented

with 20% OPFF. Extensive mycelial production of Pleurotus tuberregium on OPFF has also

been reported by Chiejina and Olufokunbi (2010). Nutritional composition of substrates is a

crucial factor in determining how mycelial growth and primordia initiation occur (Stamets,

2005). The rapidity with which L. squarrosulus developed mycelium on OPFF showed that

the waste is very suitable for its growth. This could probably be due to the residual oil and

high nutrient status of OPFF. Schisler and Patton (1971) reported that lipids are stimulatory

to mushroom growth while Okhuoya and Okogbo (1990) attributed the ability of slurry to

support the growth of mushroom to the presence of lignin, cellulose and mineral elements as

well as its residual oil. The presence of appreciable amounts of cellulose, hemicellulose and

lignin in substrates has been reported to support vigorous proliferation of mushroom mycelia.

Naraian et al. (2009) reported that mycelial growth and primordia development are dependent

on the lignocellulosic materials especially the carbon:nitrogen ratio of wastes. Our

experimental data on the chemical constituents of the organic wastes used in this study

showed that OPFF contained cellulose (74.3%), hemicellulose (12.0%) and lignin (8.50%).

The appreciable number of days to complete mycelium running on OPFF may be due to its

chemical composition and carbon:nitrogen ratio. The nitrogen content of substrates has been

shown to affect earliness of fructification and productivity of mushrooms (Philippoussis et

xli

al., 2007). The appreciable amount of nitrogen (1.36%) in OPFF when compared to the other

organic wastes explains the earliness of fructification of the mushroom on the waste. The

least time required for full mycelial colonization of OPFF may also be attributed to the waste

providing adequate aeration for the ramification of the mushroom mycelia. Substrate

structure has been reported to be an important factor for the growth of fungus mycelium as it

should be suitable for the penetration of the mycelium (Tripathy et al., 2011). Tinoco et al.

(2001) observed that the larger the surface area and pore size of substrates the more the

mycelium growth rate. Our results showed that the mushroom took the longest time for full

mycelial colonization of Gmelina sawdust (GSD). This confirms the findings of Akinmusire

et al. (2011) who obtained the longest spawn run of Pleurotus pulmonarius on sawdust

substrate. The slow growth of the mushroom on Gmelina sawdust (GSD) could probably be

due to the inability of the mushroom mycelia to produce appropriate enzymes that could

hydrolyze and convert the waste for its vegetative growth (Stamets, 2000). Mycelium

extension rate has been reported to be related to bioavailability of nitrogen in the cultivation

substrate (Philippoussis et al., 2003). The low nitrogen content (0.3%) of Gmelina sawdust

may have contributed to the slow growth of the mushroom mycelia on the waste. In addition,

chemical analyses of Gmelina sawdust showed that they contain about 8.50% hemicellulose.

Several reports have demonstrated that substrate decomposition by fungi is initially

associated to its hemicellulose content (Moyson and Verachtert, 1991). The observed slow

growth of the mushroom mycelia on Gmelina sawdust may be attributed to the low water

soluble sugar content of the waste, particularly hemicellulose when compared with the other

organic wastes evaluated. This could have exercised a negative effect during the vegetative

growth phase of the mushroom, prior to the breakdown of lignin and cellulose (Philippoussis

and Diamantopoulou, 2011). Furthermore, phytochemical studies on Gmelina genus showed

the presence of several compounds such as phenolics and iridoids (Shankar et al., 2009). The

probable presence of different phenolic substances in Gmelina sawdust (GSD) which

inhibited mushroom mycelia proliferation may have resulted to the poor mycelial

colonization of the waste. This observation agrees with the results of Winkelhausen et al.

(2005) who reported four-fold reduction of the mycelial growth rate of Alternaria solani and

Fusarium culmorum by phenolic compounds extracted from olive pomace.

The time required for mushroom primordia emergence in the present study fall within the

range reported by Oghenekaro et al. (2009) for L. squarrosulus. Our results showed that

Gmelina sawdust (GSD) and mahogany sawdust (MSD) took shorter time for primordia

initiation than oil palm empty fruit bunch (OPEFB) while oil palm fruit fibre (OPFF) took the

xlii

longest time. This result confirms the findings of Ayodele and Akpaja (2007) who found that

supplementation of sawdust with 20% OPFF advanced the time of primordia emergence of L.

squarrosulus. Kimenju et al. (2009) indicated that the time taken by the mycelia to start

pinning after ramification depends on the substrates used. The observed longest time taken

for primordia initiation of the mushroom on OPFF may be due to the high nutrient content of

the waste. Reports have shown that substrates with high quality nutrient and cellulose content

take longer time to start pinning compared to substrates with low contents of nutrient and

cellulose (Onyango et al., 2011). This is because high nutritive substrates make the mycelia

to remain vegetative for a longer period resulting in vigorous growth and late pinning

(Kimenju et al., 2009). However, other factors such as high moisture content of the substrate

have been reported to cause delayed pinning (Kimenju et al., 2009). It is worth noting that

Gmelina sawdust (GSD) showed, on the average, the slowest rate in spawn run. However, in

terms of fruiting, it took the least time for primordia emergence of the mushroom. This

probably suggests that Gmelina sawdust (GSD) may contain some kind of compound that

induces the development of the reproductive phase. It could also be due to the initial

composition of the waste, which presents a carbon:nitrogen ratio that delays spawn run but

accelerates the formation of fruit bodies (Stamets, 2000). Reports have also shown that

differences in the rates of delignification of substrates by the mushroom mycelia result in

variations in the number of days to first fruiting (Chitamba et al., 2012).

Mushroom basal growth substrates have been reported to have significant effect on

mushroom production (Mwita et al., 2011). In the present study, mahogany sawdust (MSD)

produced mushrooms with the highest fresh and dry weights. This compares favourably with

the findings of Ayodele and Okhuoya (2007) who obtained a high of yield of Psathyrella

atroumbonata on sawdust of mahogany wood. In the same vein, Joshua and Agina (2002)

reported a high yield of Pleurotus ostreatus on sawdust of Khaya ivorensis and Mansonia

altissima. Previous workers on the cultivation of mushrooms suggest that cellulose,

hemicellulose, lignin and nitrogen contents of the substrates as well as enzyme production of

the mushrooms are important criteria for yield determination (Philippoussis et al., 2003). The

observed high yield of the mushroom on mahogany sawdust (MSD) may be attributed to the

high cellulose content of 85.7% and compactness on wetting of the waste when compared to

the other organic wastes evaluated. Substrates with cellulose residues have been found to

stimulate the growth of wood rotting fungi (Fasidi and Kadiri, 1993). Also, the observed high

dry weight of mushrooms grown on mahogany sawdust (MSD) is of significance in the

mushroom preservation as it reduces the risk of contamination of the sporophores, increases

xliii

shelf life and decreases shrinking during canning ( Van Loon et al., 2000). Weight of

mushrooms whether fresh or dry is an important agronomic parameter for evaluation of the

potency of fungi as biological agents in conversion of inedible organic wastes directly into

palatable human food (Mwita et al., 2011). Therefore, the accumulation of more nutrients in

the fruit bodies measured as dry weight is of preservative and nutritional importance.

Mushrooms with the widest pileus diameter were also harvested from mahogany sawdust.

This result corroborates the findings of Kadiri and Fasidi (1990) who reported that sawdust

was consistently the best substrate for mycelial growth and fructification of fungi. According

to Duncan’s multiple range test, mean fresh weight yields of mushrooms harvested from

mahogany sawdust (MSD) and oil palm fruit fibre (OPFF) were not significantly different.

This agrees with the findings of Okhuoya and Okogbo (1991) who obtained the highest

mushroom harvest (fresh weight) of Pleurotus tuberregium on oil palm fruit fibre. Our

experimental data revealed that oil palm fruit fibre (OPFF) significantly influenced yield and

stipe height of the mushroom. This could probably be due to the high nutrient content and

residual oil of the waste. Reports have shown that developing sporophores require a supply of

lipids and proteins (ratio 50/50), that are needed for expanding cell membranes and hence

promote higher yields (Schisler, 1982). The lowest fresh and dry weights were recorded for

fruit bodies harvested from oil palm empty fruit bunch (OPEFB). This could be due to the

complex nature of the waste and/or the presence of little or no vital nutrients needed for the

mushroom growth in the substrate. The reduction in fresh weight of mushrooms has been

associated with the absence of certain specific nutrients in the substrate required by the

mushroom for its growth (Schisler, 1982). Thomas et al. (1998) also showed that complexity

of substrates impedes their efficient conversion to fungal mycelium. It is worth noting that

the mean fresh weight yields of mushrooms harvested from OPFF and OPEFB varied

significantly. This could be attributed to the complexity of the OPEFB as compared to the

OPFF. Thus, the nutrients in the fruit fibre were more accessible for the fungus than those in

the empty fruit bunch. In general, the mean yield (fresh weight) recorded in this study fall

within the range reported by Oghenekaro et al. (2009) for L. squarrosulus on sawdust of

different tropical tree species.

The efficacy of residues bioconversion process and the productivity of the mushroom crop

are assessed by the biological efficiency (BE) (Philippoussis and Diamantopoulou, 2011). In

our experiment, mahogany sawdust (MSD) produced mushrooms with the highest biological

efficiency while the least was obtained in mushrooms grown on oil palm empty fruit bunch.

There were no significant differences in BE of mushrooms harvested from mahogany

xliv

sawdust (MSD) and oil palm fruit fibre (OPFF). The observed variable ranges of BE in the

results may be due to the differences in composition and rate of degradation of the

lignocellulosic materials in the wastes by the mushroom enzymes. Variable ranges of BE

have been reported when different lignocellulosic materials were used as substrates for the

cultivation of mushrooms (Liang et al., 2009). The effects of physical and chemical

properties of substrates on yield and BE have also been investigated in Agrocybe aegerita,

Volvariella volvacea and Lentinula edodes (Philippoussis et al., 2001). Peksen and

Yakupoglu (2009) in their research reported a positive correlation among yield, nitrogen

content of substrate and BE. The biological efficiency observed in the present study compares

favourably with the findings of Ayodele et al. (2007) on L. squarrosulus grown on different

substrates.

Mushroom size is essential for its market evaluation since mushrooms with wide pilei could

be of interest in the promotion of mushroom marketing. Our results showed that mahogany

sawdust (MSD) had the highest percentage number of basidiocarps in >7 cm group hence it

produced highly marketable mushroom sizes followed by oil palm fruit fibre (OPFF).

Although the large sized fruit bodies harvested from MSD and OPFF are considered to be of

good market quality and are rated highly, Shen and Royse (2001) observed that this could be

an inferior quality since such fruit bodies tend to break during packaging thereby reducing

their quality. Gmelina sawdust (GSD) and oil palm empty fruit bunch (OPEFB) also

produced much of the mushrooms in the 3-5 cm quality group which are also marketable. No

mushrooms in 5-7 cm group were harvested from oil palm empty fruit bunch (OPEFB)

hence it did not produce highly marketable mushrooms. The differences in mushroom quality

in the different wastes could be due to the nutrient status and the nature of lignocellulose in

the respective wastes. This observation corroborates the findings of Fung et al. (2005) who

demonstrated that nutrient concentration in the substrates determined the productivity and

quality of the mushroom crop. Temperature fluctuations and accumulation of carbon dioxide

in the mushroom house during the crop cycle may also have contributed to the observed

variation in mushroom quality since environmental conditions in the room were not

efficiently controlled. Kong (2004) observed that under high carbon dioxide levels or with

less frequent ventilation, mushrooms produce long stipes with tiny caps, while they produce

short stipes with broad caps under low carbon dioxide levels or frequent ventilation.

Protein is one of the most important nutrients in food, being particularly important for

building body tissues (Quimio, 2004). In our study, harvested mushrooms were evaluated for

their nutritional status on the basis of their chemical composition. Results showed that the

xlv

highest percentage protein occurred in mushrooms grown on oil palm fruit fibre (OPFF) and

the least was observed in those harvested from mahogany sawdust (MSD). The variations in

the protein content of the mushrooms harvested from the different organic wastes may be due

to the differences in the nutritional composition of the wastes which in turn influenced the

nutritional elements available for mushroom utilization (Haq et al., 2011). This observation

agrees with the findings of Wang et al. (2001) who reported that the protein content of

mushroom is not only influenced by the protein content of the substrate but also the nature of

protein in the substrate. The mushroom mycelia are also known to secret extracellular

enzymes which play key role in the degradation of substrates and hence affect the growth,

development and nutritional value of fruiting bodies (Singh and Singh, 2011). Additionally,

the differential availability of usable nitrogen in the wastes after spawn run maybe a crucial

factor in the observed differences in the protein content of harvested mushrooms (Patil et al.,

2008). Nitrogen has been reported to be an important nutrient required for fungal growth due

to its involvement in protein, chitin and nucleic acid syntheses (Adebayo et al., 2009).

However, other factors such as the size of pileus, harvest time, stage of development and

species of mushrooms have been reported to influence the protein content of the fruit bodies

(Wani et al., 2010). The percentage protein content of cultivated mushrooms observed in the

present study fall within the range reported by Nwanze et al., (2006) for L. squarrosulus. The

protein content of L. squarrosulus has been reported to be double that of Irish potatoes and

six times that of orange (Atikpo et al., 2008). The crude protein content of this mushroom

compared favourably with and in some instances surpassed those reported for most legumes

except groundnut and soybeans grown in West Africa (Okoro and Achuba, 2012). Using this

protein content as approximate indices of nutritional quality, it would appear that this

mushroom falls between most legumes and meat. However, while the protein content of the

mushroom is still lower than that found in eggs, meat and fish, it is adequate to be used as a

substitute in the diets of the populace in the developing countries.

In conclusion, the dwindling forests in Nigeria and the absence of commercial cultivation of

L. squarrosulus has resulted to its scarcity with the few available very expensive. The

reliance on naturally growing edible mushrooms has greatly undermined the development of

mushroom cultivation to a commercial scale despite available substrate materials in Nigeria

(Okhuoya et al., 2010). The need for commercial production of some edible mushrooms in

Nigeria cannot be overemphasized in view of its potential contribution to agricultural

production and as a cheap source of protein. Tapping into the benefits of commercial

mushroom production in Nigeria will help reduce the country’s unemployment rate, increase

xlvi

her food security and revenue base while bridging her rural- urban mycophagy gap (Okhuoya

et al., 2010). The ultimate aim in the modern applied aspects of any scientific endeavour is to

integrate, wherever possible, the various disciplines of science, as well as the associated

technological processes, in order that maximum benefits may accrue from such efforts

(Chang, 2008). Combined production of mushrooms for human food, health care, animal feed

and soil conditioner/fertilizer from organic wastes should be one of the aims of such

integrated schemes that can eventually be made into profitable operations. This study

suggests that many locally available organic wastes have high potentials for utilization as

substrates for growth and production of L. squarrosulus. Our results reveal the possibility of

commercial production of high quality L. squarrosulus on oil palm fruit fibre (OPFF) and

mahogany sawdust while OPFF is recommended as the best substrate for spawn production

among the various organic wastes evaluated. Despite differences in the protein content of

mushrooms grown on the different organic wastes evaluated, the overall nutritional potential

of the mushroom is quite good. As a result of the low values recorded for biological

efficiency (BE) in this experiment, there is need for supplementation of these wastes with

nitrogen and carbohydrate based additives or supplements in order to enhance better BE and

yield of the mushroom.

xlvii

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