CHAPTER ONE
1. Introduction
1.1. Background
Economies of countries across Africa including Ethiopia have a particularly
high reliance on agriculture. However, malnutrition and food insecurity
continue to be major concerns in the continent (Albanie et al., 2021). The
escalating impacts of global warming are also placing the agriculture sector
at significant risk (Chandio et al., 2020). Mushroom cultivation could play a
vital role in meeting the demands of food scarcity by sustaining food
production and security (Niazi & Ghafoor, 2021). From very early times
humans have collected wild mushrooms for food, and it is estimated that the
first intentional cultivation of mushrooms took place around 600 A.D. Since
then, the cultivation of edible mushrooms has increased significantly (Miles &
Chang, 1997).
A mushroom is a type of macrofungus that possesses a distinctive fruiting
body. These fruiting bodies can either emerge above ground (known as
epigeous) or remain underground (referred to as hypogenous). They are
visible to the naked eye and can be harvested by hand. Mushrooms are
commonly found in fungi of the class Basidiomycetes and occasionally in the
class Ascomycetes (Cheung, 2008). Mushrooms are a fascinating component
of the human diet, offering both nutritional and therapeutic properties. While
some mushrooms are celebrated worldwide for their health benefits, others
can be perilous due to their toxicity (Jo et al., 2014).The earliest recorded
evidence of mushroom cultivation hails from China, where farmers began
growing shiitake mushrooms over 1,000 years ago. However, the practice of
cultivating mushrooms for food likely began even earlier, around 600 AD
(Miles & Chang, 1997). Among commercially cultivated mushrooms, Oyster
mushrooms, belonging to the genus Pleurotus, are widely recognized as
valuable edible fungi and are extensively cultivated worldwide. These
mushrooms are prized for their high nutritional value, delicate flavor, and
varying vitamin and mineral content (Bulam et al., 2019). Oyster mushroom
cultivation requires simple and straightforward cultivation techniques whilst
exhibiting rapid fruiting and high biological efficiency. According to Cheung
(2008) in terms of industrial cultivation, oyster mushrooms rank second
globally, following the white button mushroom (Agaricus bisporus). Since
they require no arable land for production, they are a suitable option for all
mushroom growers. It is also an excellent venture for those mushroom
growers that are residing in urban areas (Kratika, 2018).
1.2. Statement of the Problem
Malnutrition and food insecurity pose significant challenges in Ethiopia.
Traditional methods of farming also contribute to this problem. Given the
ongoing global climate crisis, it is becoming more difficult to cultivate
common cash crops. Despite the growing interest in mushroom cultivation,
there is a lack of comprehensive cultivation technique. Understanding the
specific challenges, optimal substrates, and growth parameters for oyster
mushrooms in this context remains essential. Therefore, this research aims
to develop effective strategies for the cultivation of oyster mushrooms under
laboratory and greenhouse conditions, with a focus on optimizing growth
parameters, producing high quality spawn, and elucidating the potential
medicinal properties of the cultivated mushrooms.
1.3. Significance of the Study
Despite the growing interest in mushroom cultivation, there is a lack of
comprehensive research on oyster mushroom cultivation under laboratory
conditions in Addis Ababa, Ethiopia. Understanding the specific challenges,
optimal substrates, and growth parameters for oyster mushrooms in this
context remains essential. Therefore, this research aims to develop effective
strategies for the cultivation of oyster mushrooms under laboratory
conditions, with a focus on optimizing growth parameters, assessing
nutritional attributes, and elucidating the potential medicinal properties of
the cultivated mushrooms.
CHAPTER TWO
2. Literature Review
2.1. An Overview of Mushrooms
Mushrooms belong to the kingdom Fungi, which is a distinct group separate
from plants, animals, and bacteria. Unlike plants, fungi do not possess the
ability to directly harness energy from the sun through chlorophyll. As a
result, fungi rely on other organisms for sustenance, obtaining nutrients from
the organic material in their surroundings. They can function as
decomposers, symbionts, or parasites. The living part of the fungus is called
mycelium and is composed of tiny threads known as hyphae. Under specific
conditions, sexually compatible hyphae merge and begin to produce spores.
The mushroom, which is a larger structure (typically larger than 1 mm)
responsible for spore production, is often the most visually striking part of
the organism in nature. However, it is important to note that the mushroom
is simply the fruiting body of the fungus (Wasser, 2006).
Oyster mushrooms, belonging to the Pleurotus genus, are highly regarded
edible fungi that have attracted considerable attention in recent years due to
their nutritional value, culinary versatility, and potential health benefits
(Chang & Miles, 2004). These mushrooms, characterized by their oyster
shell-shaped caps, are widely distributed across the globe, with various
species found in different regions and climates. Several species within the
Pleurotus genus are commonly cultivated, each with its own unique
characteristics and cultivation requirements. There are many different
species of Oyster mushrooms. Some of these include: Pleurotus sajor-caju,
Pleurotus membranaceous, Pleurotus sapidus, Pleurotus florida, Pleurotus
ostreatus, Pleurotus eous, Pleurotus flabellatus. Among the different species
of Pleurotus, Pleurotus ostreatus is the most cultivated due to it short growth
period, fewer environmental demands and resistance to disease and pests
(Cheung, 2008). Pleurotus ostreatus, also known as the pearl oyster
mushroom, has a gray to dark brown cap with a smooth or slightly wrinkled
surface. This species is known for its adaptability to a wide range of
substrates, including agricultural wastes such as straw, sawdust, and corn
cobs (Bellettini et al., 2019).
2.1.1. Poisonous and Edible Mushrooms
Mushrooms have captivated human interest for centuries due to their diverse
properties, ranging from culinary delights to potential health benefits. The
classification into poisonous and edible mushrooms is crucial for safe
consumption. Reports have extensively documented the medicinal and/or
toxic effects of various fungal species. Unfortunately, cases of serious human
poisoning often result from improper identification of toxic mushroom species
(Jo et al., 2014). Different substances responsible for the fatal signs and
symptoms of mushroom toxicity have been identified from various poisonous
mushrooms. For instance, the Amanita phalloides (death cap) contains
deadly toxins. Toxicity studies have shown that mushroom poisoning can lead
to adverse effects such as liver failure, bradycardia, chest pain, seizures,
gastroenteritis, intestinal fibrosis, renal failure, and even death (Verma et al.,
2014).
Some important distinctions between poisonous and non-poisonous
mushrooms are as follows. Although these distinctions are not completely
reliable, they can be used for identification to prevent accidental deaths
caused by consuming poisonous mushrooms. According to Hall et al. (2010)
one distinctive feature of poisonous mushrooms is the presence of a ring or
annulus in the middle of the stalk or stipe. Another characteristic feature is
the presence of a cup or saucer-like structure called a volva at the base of
the stipe. Generally, both of these structures are found together in poisonous
mushrooms. However, in edible species, either the ring (e.g. Button
mushroom) or the volva (e.g. Paddy straw mushroom) may be present, or
neither of these structures may be present (e.g. Oyster mushroom).
Poisonous mushrooms are relatively soft and the skin of the cap cannot be
easily peeled off. They are usually colorful and attractive. Some poisonous
mushroom species exude a milk-like substance when their fruit bodies are
damaged. Poisonous mushrooms often have a bitter or sour taste and emit
an unpleasant smell.
2.1.2. Nutritional and Medicinal Aspects of Oyster Mushrooms
The cultivation of oyster mushrooms has increased globally due to their
attractive nutritional profile and medicinal properties. Oyster mushrooms are
low in calories and fat, making them a healthy addition to meals. They are
rich in protein, fiber, and vitamins, including B-vitamins (such as B1, B2, B3,
B5, and B6). These mushrooms also contain essential minerals like iron,
calcium, and potassium. Oysters have several antioxidants and possess
hypocholesterolemic and hypoglycemic properties helping regulate
cholesterol and glucose levels respectively. They also exhibit anti-
inflammatory properties and may help enhance immune function. Oyster
mushrooms contain compounds like ergothioneine and glutathione, which
have antioxidant properties. (Bulam et al., 2019). In addition they are a
culinary delight in many countries due to their unique texture and flavor.
2.2. Utilization of Ligninocellulolytic Wastes
Oyster mushrooms secrete different enzymes that are capable of breaking
down the carbohydrates and lignin found in several plant residues enabling
them to grow over a wide variety of substrates (Sözbir et al., 2015).
Cultivating mushrooms on organic wastes is a sustainable way of recycling
nutrients whilst still producing a valuable output. The substrates used to
cultivate this mushroom variety can include industrial wastes such as wood
shavings, various types of straw, as well as other plant waste materials such
as coffee pulp (Sharma et al., 2021). Some studies also show that oysters
can be cultivated on certain plant weeds thus providing an alternative
solution to the problem that plant weeds pose (Ejigu et al., 2022). Numerous
investigations have been carried out to determine the optimal substrate type
as well as the effect of different substrate combinations for oyster mushroom
cultivation, taking into account factors such as growth, yield, and biological
efficiency (Sardar et al., 2020). Pleurotus ostreatus has the ability to adapt
the expression of its lignocellulolytic enzymes with respect to the different
lignin and carbohydrate composition of its substrates making it an excellent
choice for the utilization of several agricultural wastes (Xiao et al., 2019).
2.3. Cultivation Techniques
The availability of good quality spawn is the limiting factor for mushroom
cultivation in many developing countries. Spawn production is involves
putting mycelium of the desired mushroom in suitable sterilized substrates
under sterile conditions. The first steps in spawn production are performed
on artificial media like PDA or MDA. The mycelium grows on the surface of
the medium and will later be used to inoculate larger amounts of substrates
like sawdust or cereal grain (Gume et al., 2013).
Agricultural waste such as wood chips/sawdust, sugar cane bagasse, and
various types of straw can serve as the primary components of the substrate
for oyster mushrooms. The properties of the substrate determine the types
of mushrooms and microbes that can thrive in it. The selectivity of the
substrate dictates how well it caters to the specific requirements of a
particular mushroom, making it less suitable for others. Once the substrate is
mixed and supplemented, it undergoes a heat treatment to create an
environment that minimizes competition for the desired mushroom mycelium
(Fanadzo et al., 2010).
Multiple techniques exist for growing oyster mushrooms. Certain methods
involve cultivating them on cut wood logs, while others employ culture media
housed in jars or plastic bags. Alternatively, some growers choose to place
the culture substrates on surfaces like ridges, bed frames, or within variously
sized containers (Chang & Miles, 2004).
CHAPTER THREE
3. Research Questions, Hypothesis and Objectives
3.1. Research Questions
What methods can be employed to produce high-quality oyster
mushroom spawn?
What are the economic implications of oyster mushroom cultivation in
a laboratory?
Can this method be scaled up for commercial production, and what
challenges might arise?
3.2. Research Hypothesis
Hypothesis 1
A high-quality oyster mushroom spawn can be prepared inside a laboratory
following strict sterilization procedures.
Hypothesis 2
Cottonseed hulls are an effective substrate for the cultivation of Pleurotus
Ostreatus.
3.3. Research Objectives
3.3.1. General Objective
• This research aims to explore the biological efficiency and yield of
oyster mushrooms (specifically Pleurotus Ostreatus) cultivated under
laboratory conditions.
3.3.2. Specific Objectives
Assess spawn quality based on mycelial growth rate, vigor, and
absence of contaminants.
Analyze the costs associated with substrate, spawn production,
utilities, and labor to determine their respective profitability.
Investigate scalability mechanisms by addressing challenges related to
space and labor.
CHAPTER FOUR
4. Materials and Methods:
This research was conducted at Addis Ababa University, Department of
Microbial, Cellular and Molecular Biology. Given below is a list of the
chemicals, tools and instruments used to conduct this research.
4.1. Materials
4.1.1. Chemicals:
Potato Dextrose Agar, ethanol, distilled water, sorghum grains, gypsum,
wheat bran, cottonseed hulls.
4.1.2. Tools/ Instruments:
Petri dishes, beaker, weight balance, spatula, inoculating loop, glass bottles,
plastic bags, polypropylene bags, laminar flow hood, incubator and
autoclave.
4.2. Methods
4.2.1. Laboratory and Greenhouse (Mushroom Farm) Setup
Laminar flow hood and incubator present in the laboratory were sterilized
using alcohol. Floors, walls and tables inside the greenhouse were cleaned
thoroughly. A greenhouse room was also cleaned thoroughly and prepared in
such a way that pests aren’t able to get inside. Separate wooden logs were
arranged on tables about half a meter high.
4.2.2. Mushroom Mycelial Culture Preparation
The mycelial growth of oyster mushrooms was isolated and purified from the
stipe or stem of these mushrooms previously grown in the Mycology
laboratory and greenhouse of the Department of Microbial, Cellular, and
Molecular Biology of the College of Natural and Computational Sciences at
Addis Ababa University. This mycelial culture was sub-cultured on an agar
medium. To prepare the culture medium 9g of Potato Dextrose Agar (PDA)
powder was mixed with 230ml of distilled water in a beaker and autoclaved
at 121ºC for 30 minutes. The PDA solution was poured into 10 Petri dishes,
and left to solidify inside a hood. In a sterile environment (laminar flow
hood), a small piece of oyster mushroom mycelium was taken from the
mother mycelium culture and transferred onto the surface of the agar
medium using a sterile inoculating loop. The inoculated cultures were
incubated at normal room temperature until the medium was fully colonized
with the mycelium.
Figure 1: PDA Preparation
4.2.3. Spawn Preparation
A suitable carrier material was prepared by washing 1kg of sorghum grains
to remove any impurities. It was then soaked in water for 24 hours. The next
day, the grains were drained and mixed with 2% gypsum on a dry weight
basis (i.e. 20gm) and 15% wheat bran (i.e. 150gm) in order to adjust the pH
and nitrogen levels, respectively. The mixture was poured into ten glass
bottles and put inside the autoclave at 121ºC for 30 minutes. The mycelium
culture inside the petri dishes was cut into smaller pieces and used to
inoculate the sorghum grains. Each bottle was inoculated by one petri dish.
The spawn mixture was incubated at an optimal temperature (around 25°C)
until fully colonized. The bottles were then stored in a cool and dark place
until they were ready to be used.
Figure 2: Sorghum grains preparation
4.2.4. Substrate Preparation and Inoculation
Cottonseed hulls were selected as a suitable substrate for oyster mushroom
cultivation. 5kg of the substrate was soaked in water for hydration. The
excess water was squeezed by hand to get the mixture to an appropriate
moisture level, roughly around 60%. 2% gypsum (i.e. 100gm) and 15%
wheat bran (i.e. 750gm) were added. The mixture was poured into plastic
bags and autoclaved at 121ºC for 30 minutes. The substrate was transferred
into six polypropylene bags, each weighing around 0.9kg. Each bag was
inoculated with 300gm of spawn. The spawn and substrate were mixed
thoroughly. The bags were plugged with cotton to allow for proper oxygen
exchange.
Figure 3: Substrate (cottonseed hulls) preparation
4.2.5. Cropping and Harvest
The bags were transferred into a dark greenhouse room that doesn’t get any
direct sunlight and put on wooden logs within 20cm of each other. The
growth and development of the mushrooms were monitored i.e. the
mycelium colonization progress was observed within a one day interval.
When the spawn run was complete and pinheads started to emerge the
plastic bags were split open at appropriate sites and then watered
continuously on a daily basis. In order to help increase the humidity of the
room, the wooden tables were also wetted. When the pinheads developed
into mature fruit bodies, they were harvested by gently twisting the stalk at
its base, where it was attached to the substrate.
Figure 4: Cultivation and harvest
CHAPTER FIVE
5. Results and Discussion
Oyster mushroom (Pleurotus ostreatus) was grown on cottonseed hulls
supplemented with wheat bran as substrate. The growth and yield of
Pleurotus ostreatus on six identical treatments is depicted in Table 1 and
Table 2.
5.1. Mycelial Culture Preparation
The mother mycelial culture was used to inoculate 10 agar-filled petri dishes.
Total colonization of the medium with the mushroom mycelium took 7 days
and the cultures were successfully obtained.
Figure 5: Inoculation with mother mycelium culture
5.2. Spawn Preparation
After the sorghum grains were inoculated with the mushroom mycelium it
took 7 days for the mycelium to fully colonize the substrate. During the
spawn production process, some of the bottles were contaminated. The ones
that were contaminated were discarded and a new batch was made. The
contamination could have been due to properly unsterilized equipment or
improper handling and storage of the spawn.
Figure 6: Spawn incubation and final result
5.3. Spawn Running Stage
Cotton seed hulls were inoculated with the mushroom spawn and transferred
to the greenhouse. After 24 hours of spawning, mycelial extension was
observed in the substrate. Total colonization of the substrate happened on
the 15th day after inoculation. This goes in line with the findings of Shnyreva
et al. (2017) which showed it took 17 days for spawn run.
Figure 7: Substrate inoculation to complete spawn run
5.4. Pinhead Formation
The pinhead formation was observed between 9-12 days for all treatments
after a period of complete mycelial colonization. The time taken for
primordial formation after inoculation was recorded to be 24-27 days. After
complete mycelial colonization the mushrooms weren’t transferred to a
different cropping room for primordial formation and maturation of fruit
bodies. This could have increased the time needed to induce pinhead
formation and the subsequent maturation of fruit bodies.
Figure 8: Complete spawn run to pinhead formation
5.5. Fruit Body Formation
The number of days taken from pinhead formation to the development of
fruiting bodies ranged from 4-6 days. The time taken for the fruiting body
formation of the mushroom after inoculation on different substrates was
recorded to be between 28-33 days. The second flush occurred around 38-43
days after inoculation while the third flush took about 50-55 days after
inoculation. The 1st, 2nd and 3rd flushes occurred within a span of 10-12
days.
Figure 9: Fruit body formation
Commencing from the day of substrate (cottonseed hull) inoculation, the
time required for the complete spawn run, pinhead formation, subsequent
development of mature fruit bodies and 1st to 3rd flush is presented in the
table below.
Table 1: Time required for the complete spawn run, pinhead formation,
development of mature fruit bodies and 1st to 3rd flush
No Activities Dates of mushroom growth observation on average
1. To complete spawn run 15 days
2. For pinhead formation 25 days
3. For development of matured fruit bodies 30 days
4. 1st harvesting of matured mushrooms 32 days
5. 2nd harvesting of matured mushrooms 42 days
6. 3rd harvesting of matured mushrooms 54 days
5.6. Fresh Weight of Fruiting Bodies
After the formation of mature fruit bodies the mushrooms were harvested by
gently twisting the stalk at its base, where it was attached to the substrate.
The greatest (2.25kg) weight of harvested mushrooms was recorded during
the first flush and the lowest (1.51kg) was recorded during the third flush.
During the second harvest, a total of 1.93kg of mushroom fruit bodies were
harvested.
Figure 10: Weight of fruit bodies
Table 2: The fresh weight of oyster mushroom (gm) for the 1st, 2nd and 3rd
harvests.
No.
Types of harvest of matured mushroom
Fresh weight of mushroom (gram)
1 1st harvest 750
500
650
350
Total 2250
2 2nd harvest 600
700
630
Total 1930
3 3rd harvest 600
510
400
Total 1510
The yield of the oyster mushrooms varied across six different treatments
throughout the entire harvesting period. These results clearly indicate that
the fluctuations in oyster mushroom yield are due to the efficient utilization
of nutrients from the substrate, which consists of cotton seed hulls. The
utilization of nutrients across each treatment is dependent on the moisture
content within the bags. There also seems to be a correlation between the
size of the holes and diameter of the pileus, with larger holes bearing larger
fruit bodies than the smaller holes.
5.7. Biological Efficiency
The potential for using cottonseed hulls supplemented with 15% wheat bran
as essential substances for substrate preparation in the cultivation of oyster
mushrooms (Pleurotus ostreatus) and their effects on the yield and biological
efficiency (BE) were determined. The results indicated that cottonseed hulls
could be used as very good materials in the preparation of substrates for the
cultivation of Pleurotus ostreatus.
Biological efficiency (BE) for the three different flushes was calculated using
the following formula:
% BE = Total weight of fresh mushroom x 100 %
Dry weight of substrate
A total of 2.25kg, 1.93kg and 1.51kg were harvested during the 1st, 2nd and
3rd flushes respectively. Biological efficiency for each harvest was calculated
as follows.
1st harvest: % BE = 2.25 kg x 100% = 45 %
5kg
2nd harvest: % BE = 1.93 kg x 100% = 38.6 %
5kg
3rd harvest: % BE = 2.25kg x 100% = 30.2 %
5kg
Figure 11: Graphical representation of the biological efficiency of the three
harvests
The total biological efficiency, taking into account all three harvests
combined, was calculated to be 113.8%. Similarly, Yang et al. (2013) found
that cottonseed hulls supplemented with 20% wheat bran resulted in 125.6%
BE. Taking into account factors such as the costs associated with substrate,
spawn production, utilities, and labor their respective profitability was
calculated at 20% profit margins. Therefore, the results of this research
suggest that cottonseed hulls are indeed an efficient substrate material for
the cultivation of Pleurotus ostreatus.
Figure 12: Harvest period
CHAPTER SIX
6. Conclusion and Recommendations
6.1. Conclusion
A high-quality spawn can be produced inside the laboratory whilst following
strict sterilization techniques. Cottonseed hulls are indeed a suitable
substrate for cultivating Pleurotus ostreatus. Cultivating Pleurotus ostreatus,
offers several benefits when done in both laboratory and greenhouse
settings. Laboratory and greenhouses allow precise regulation of
temperature, humidity, and light, promoting optimal mushroom growth. In
addition, Greenhouses accommodate larger cultivation areas, enabling
commercial production. Even though Pleurotus ostreatus are referred to as
winter oyster mushrooms, they have very versatile growth requirements
unlike most commercial crops and can grow under stressful environmental
conditions. Therefore, it is possible to conclude from this research that
Pleurotus ostreatus can be successfully cultivated on cottonseed hulls under
laboratory and greenhouse conditions.
6.2. Recommendations
Further research could explore optimizing greenhouse conditions for
Pleurotus species, including substrate composition, air exchange, and light
exposure. Additionally, investigating the impact of different environmental
factors on fruit body quality and yield would enhance our understanding of
oyster mushroom cultivation.
In conclusion, combining laboratory insights with greenhouse scalability
offers a comprehensive approach to cultivating oyster mushrooms. By
leveraging both settings, we can meet the growing demand for these
nutritious and flavorful fungi.
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