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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
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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.
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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…………………………………
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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).
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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
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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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