LIKHA PROPOSAL

(1) PROJECT PROFILE

Project Title: Mycoremediation potential of paddy straw mushroom ( Volvariella

volvacea) for oil-contaminated soil

Name of Project Proponent/s:

Yuri Gabrielle A. Bona

Anthea Florence B. Portacio

Kaecy Nicole B. Velitario

Region: V- BICOL Division: Camarines Sur

School: Nabua National High School Grade Level: Grade 9

Project Duration (Number of months): Three (3) months

Email: bonayuri15@gmail.com Contact Number: 09175905763

antheaportacio@gmail.com 09303210991

kaecyvelitario@gmail.com 09054140630

(2) CATEGORY OF RESEARCH (4) THEME

________ Physical Science ______ Food Safety

________ Life Science ______ Water Conservation

________ Robotics and Intelligence ______ Renewable Energy

________ Mathematics and ______ Cyber Security

Computational Sciences ______ Traffic/Road Congestion

______ Health

(3) ______ Disaster Mitigation

________Individual ______ Agriculture and Environment

________Team ______ Others (please specify)

_____________________________

(5) EXECUTIVE SUMMARY

          What runs the world can ruin it. Oil, as the major source of energy in the world

since the mid-1950s, is the essential constituent in the transport, construction, and

agriculture sectors. When accidents related to oil happen, however, it can create

great damage to our environment, including a significant decline in biodiversity and

life itself. From the year 2020 to 2021, oil spillages increased with a large spill in Asia

and five other medium spills recorded. With these adversities, researches on the most

effective treatment for soil contamination due to oil spillage are greatly essential.

          The present research focuses on reviewing the potential of the paddy straw

mushroom (Volvariella volvacea) for mycoremediation of oil-contaminated soil since it

thrives easily in the climate of the Philippines and has specific enzymes that can

break down the components of the oil.

          The research study will test the null hypothesis that there is no significant
difference between the 3 treatments. In terms of: a. mushroom growth, b.growth

inhibition of the soil as an indication of its toxicity, and c. soil quality of the blocks

using the different treatments.

          Each type of contaminated soil will be replicated thrice and be randomly treated

with different treatments. The researchers will make use of a statistical test for data

analysis. The soil will be further tested by planting pechay (Brassica rapa) seeds.

(6) INTRODUCTION

(6.1) RATIONAL/SIGNIFICANCE

Fungal bioremediation is the most environmentally friendly and sustainable

method of cleaning up polluted sites. Fungi have a variety of methods for eliminating

various toxins and recalcitrant contaminants, including the secretion of powerful

fungal enzymes. Bioremediation is a cost-effective and environmentally friendly

process that uses several biological methods to convert recalcitrant contaminants into

environmentally benign products. Fungi play critical roles in bioremediation due to

their robust morphology and diverse metabolic capacities. Bioremediation is a low-

cost, environmentally friendly method of converting toxic, recalcitrant pollutants into

environmentally benign products using various biological treatments. Because of their

robust morphology and diverse metabolic capacity, fungi play an important role in

bioremediation. Mushroom has been used for consumption as a product for a long

time due to their flavor and richness in protein. Mushrooms are also known as

mycoremediation tools because of their use in the remediation of different types of

pollutants. Mycoremediation relies on the efficient enzymes, produced by

mushrooms, for the degradation of various types of substrate and pollutants. Besides

waste degradation, mushrooms produced a vendible product for consumption.

However, sometimes they absorb the pollutant in their mycelium (biosorption process)

and cannot be consumed due to absorbed toxicants.

Volvariella volvacea is the fifth most important edible mushroom in the world

according to yield( Chang 1993). It is a tropical and subtropical saprophytic fungus

belonging to the family Pluteaceae of Basidiomycetes. It is commonly known as

paddy straw mushroom, Chinese mushroom, and tributary mushroom. Volvariella

volvacea is also known for its unique aroma and texture. The nutritional value of

these mushrooms depends on the type of agricultural waste used for its production

(Roy et al., 2014). Enzymes occur in all living organisms including mushroom and it is

used for hydrolysis; oxidation, reduction, or metabolism (Quimio, 1989; Wang, 1989).

It plays a vital role in the mushroom development, nutritive value and flavour

(Jonathan, 2002). The enzymes like amylase and cellulase from various fungal

sources has largely been screened for commercial utility (Wang, 1989; Diez and

Alvarez, 2001). Celluloytic enzymes play a significant role in natural biodegradation

process (Jonathan and Adeoyo, 2011).

Leakage of oil and its derivatives into the soil can change the engineering

behavior of soil as well as cause environmental disasters. Also, recovering the

contaminated sites into their natural condition and making contaminated materials

both environmentally and geotechnically suitable construction materials need the

employment of remediation techniques. Bioremediation is an efficient, low cost and

environmental-friendly approach. Mycoremediation is a process that is used to

degrade or isolate contaminants in soil. It is used in soil contaminated with petroleum

or diesel oil as the fungi can reduce the polycyclic aromatic hydrocarbons (PAH)

(Shaw and Brink, 2011). Mycoremediation is an environmentally friendly alternative

technique for contamination remediation in environmental matrices. It employs fungi

and has been used on both soil and water. The technique has several advantages

over other bioremediation, physical, and chemical methods. Aside from cost and

technical ease, the ubiquitous nature of most fungi species could allow for

widespread applications in various parts of the world. According to Leo-nardi and

Rahman, mycoremediation appears to be the safest method of soil remediation in

terms of ecological impact and human health. Because most organic contaminants

are degraded rather than extracted, the risk of bioaccumulation and pollution transfer

into the food chain is reduced.

This current study investigates the effect of paddy straw mushrooms on oil-

contaminated soils.  This study will be used as a reference by future researchers with

the purpose of enhancing research. It aids in developing new diagnostic tests,

treatments, and processes that may one day benefit other researchers. This study will

have many benefactors such as animals, humans, and the environment itself. This

study aims to lessen pollution that can be harmful to the environment and humans.

The benefit of this research will be helpful to everyone for it will lessen the harm of

exposure to PAHs that may cause increased incidences of lung, skin, and bladder

cancers due to occupational exposure to PAHs. In this case, farmers and foresters

working near oil factories would benefit from this study. This way, they would be
aware of the methods they can use and make the correct measurements to lessen

their exposure to PAHs.

(6.2) SCIENTIFIC BASIS/THEORETICAL FRAMEWORK/MATHEMATICAL

THEORY

Soils contaminated with toxic and persistent pollutants pose different and

serious hazards to the environment and human health. Soil pollution can be

generated by the presence of human-made chemicals or other changes in the natural

soil environment (Diana Mariana Cocâr¸tă et. al. [2017]). Specifically, this could be

caused by industrial activities, agricultural chemicals, or inappropriate disposal of

waste. According to European Environmental Agency (EEA) studies on the basis of

non-harmonized national inventories, there are 2.5 million potentially contaminated

sites in Europe (EEA-33 plus the six cooperating countries). About one-third (an

estimated total of 342,000 contaminated sites) have already been identified and about

15% of these have been remediated (Aykan Karademir et. al. [2017]).

In relation to all of this, the main aim of this research is to create a solution in

oil-contaminated soil using Volvariella volvacea (Paddy Straw Mushrooms). Wherein

it also aims to ease the damage of soil pollution, severe hazards to the environment,

and human health.

Volvariella volvacea is also known as "the warm mushroom" as it grows at a

relatively high temperature. The optimum temperature and relative humidity for the

growth of this mushroom are 30–35°C and 80–90%, respectively. It is a fast-growing

mushroom and, under favorable growing conditions, the total crop cycle is completed
within 3–4 weeks.

Paddy straw mushroom can use a wide range of cellulosic materials and the C:

N ratio needed is 40 to 60, quite high in comparison to other cultivated mushrooms. It

can be grown quite quickly and easily on uncomposted substrates such as paddy

straw, cotton waste, or other cellulosic organic waste materials (Ahlawat & Kumar,

2005). It has been considered as one of the easiest mushrooms to cultivate.

Bioremediation is a process that can degrade various pollutants,organic and

inorganic, using microbial organisms such as fungi, bacteria, etc. Bioremediation by

microbes can be in situ or augmented by external introduction. Extensive studies

conducted on edible mushrooms and other white rot fungi have proved their inherent

capacity to biodegrade xenobiotic compounds, which otherwise take quite a long time

for their complete mineralization. This bioremediation employing mushrooms and

other white rot fungi is gaining wide popularity as a mycoremediation technique.

Mycoremediation has a versatile role in environmental protection.

Mycoremediation is an eco-friendly, noninvasive, cheaper, and easier solution that

can reduce or transform environmental hazards into non-toxic or harmless forms

(Perelo, 2010).

Volvariella volvacea including other mushrooms, produced  enzymes like

amylase and cellulase which plays a vital role in the mushroom development

(Jonathan, 2002), and natural biodegration process (Adeoyo, 2011).

In a study by Karnan, et al. (2016), the screening and biochemical

determination showed presence of saponin, alkaloids, terpenoids, sugar, flavonoids

and sterols. Amylase, cellulase and laccase are important enzymes that can be used
for various biological activities. Laccase exhibit good enzyme production than other

enzyme like amylase and cellulase. This investigation may provide a basic knowledge

about the Volvariella volvacea, and give valuable information for further study. In this

study, the filtrates of each paddy straw mushroom were assayed for Cellulase using

the modified dinitrosalicylic acid (DNSA) reagent method of Zhou et al., (2009). The

amount of reducing sugar that was released was determined by adding 1 mL of

DNSA to 1 mL of filtrate-starch-reaction mixture, and the absorbance was read at 540

nm using a spectrophotometer. Cellulase activity in the filtrate was determined by the

method of (Zhou et al., 2009). The assay medium contained 0.55% carboxymethyl

cellulose (CMC) in 0.55M acetate buffer (pH 6.8), and the reducing sugars released

were measured by the DNSA reagent method of (Parra et al., 2005).

(6.3)

OBJECTIVES

GENERAL: This study generally aims to review the potential of paddy straw

Mushroom (Volvariella volvacea) for oil-contaminated soil degradation.

SPECIFIC:

(1) Determine the mean mushroom growth of the different contaminated

soil in the different treatments: a. Treatment 1 (oyster mushroom (Pleurotus

ostreatus) mycelium, b. Treatment 2 (paddy straw mushroom (Volvariella

volvacea) mycelium), and c. Treatment 3 (control with no treatment).

(2) Determine the mean growth inhibition of the soil as an indication of

its toxicity of the different contaminated soil in the different treatments: a.

Treatment 1 (oyster mushroom (Pleurotus ostreatus) mycelium, b. Treatment 2

 (paddy straw mushroom (Volvariella volvacea) mycelium), and c. Treatment 3

(control with no treatment).

(3) Determine the mean soil quality of the different contaminated soil in

the different treatments: a. Treatment 1 (oyster mushroom (Pleurotus

ostreatus) mycelium, b. Treatment 2 (paddy straw mushroom (Volvariella

volvacea) mycelium), and c. Treatment 3 (control with no treatment).

(4) Determine if there is a significant difference among the

mycoremediation potential of the three different treatments in terms of: a.

mushroom growth, b. growth inhibition of the soil as an indication of its toxicity,

and c. soil quality.

(7) REVIEW OF RELATED LITERATURE

The world uses oil in almost every aspect of life. Environmental Pollution

Centers stated in 2022 that when an oil spill occurs, many elements of the

environment may be affected.

On Oil Contamination

Oil is the dynamic force of industrialized countries as it is one of the major

sources of energy. Oil spills are defined as leakage and release of oil from petroleum

extraction, storage, distribution, and refinement sites into the environment, which can

threaten the marine, coastal, and land ecosystem. Oil spills have disastrous impacts

on society, economy, and environment (Broekema, 2016). When soil becomes

contaminated with oil due to these oil spillages, it becomes toxic. Withal, oil

contamination does not only harm the soil itself; it also creates drastic changes to

biodiversity and further community pollution. Deepwater Horizon oil spoil in 2010 and
Exxon Valdez oil spoil in 1989 are the major oil spills with the most severe

environmental impacts (Salimnezhad, et al., 2021).

Soil contamination, as one of the most critical cases of contamination, not only

changes the chemical, physical and biological properties of soil but also affects the

geotechnical properties of soil (Salimnezhad, et al., 2021). Oil-contaminated soil can

be treated with biological, physical, or thermal methods. Examples of biological

methods are bioremediation; mycoremediation is a form of bioremediation.

On Mycoremediation

Mushroom has been used for consumption as product for a long time due to

their flavor and richness in protein. Mushrooms are also known as mycoremediation

tool because of their use in remediation of different types of pollutants.

Mycoremediation is based on the use of fungi and mushroom for the removal of

waste from the environment. It is a form of bioremediation. The mushrooms and other

fungi possess enzymatic machinery for the degradation of waste/pollutants and

therefore, can be applied for a wide variety of pollutants (Purnomo et al., 2013;

Kulshreshtha et al., 2013).

Mycoremediation relies on the efficient enzymes, produced by mushroom, for

the degradation of various types of substrate and pollutants. (Kulshreshtha, et al.,

2014). Mycoremediation tool refers to mushrooms and their enzymes due to having

the ability to degrade a wide variety of environmentally persistent pollutants and

transform industrial and agro-industrial wastes into products.

On Paddy Straw Mushrooms

Paddy straw mushroom is a native species to East Asia, and it is very popular

in Asian cuisine. It is a truly a tropical mushroom, and perfect for intense summer

heat. These mushrooms fruit only above 80F and actually prefers 90+F, this protein

rich species can be grown on many other slightly composted, dried vegetable wastes.

Paddy straw mushroom is having good combinations of all attributes like flavour,

aroma, delicacy, high content of protein and vitamins and minerals, because of which,

the acceptability of this mushroom is no way less than much popular white button

mushroom. It is an edible mushroom of the topics and subtropics, and began to be

cultivated in China as early as 1822. Around 1932-, the straw mushroom was

introduced into the Philippines, Malaysia, and other south-east Asian countries by

overseas Chinese (DMR, Solan, HP). Since then, its cultivation has been conducted

in various countries outside of the region. The fruiting body formation starts with tiny

clusters of white hyphal aggregates called primordia and it is followed by several

morphological stages in the fruiting body development process. The successive

stages are called as "button", "eggs", "elongation", "mature" stages respectively.

Differentiation can be seen first at the 'button' stage. At maturity the buttons enlarge

and umbrella like fruit bodies emerge after the rupture of the volva (DMR, Solan, HP).

Volvariella volvacea is a typical edible edible straw mushroom with a high-

temperature tolerance that preferentially grows at 30°C. It has a strong fibrinolysis

capability and consumes abundant agricultural wastes. Its fruiting bodies are popular
with consumers owing to their taste and high nutrient contents. Moreover, V. volvacea

contains many bioactive substances with medicinal values, such as anticancer-

associated polysaccharides, immunosuppressive proteins, and immunoregulation-

associated agglutinins (Mathew et al., 2008; Wu et al., 2011; Sun et al., 2014).

On Oyster Mushroom

Akkin N. et al (2021) mentioned that oyster mushroom is the Pleurotus species,

which is a wellknown fungus that can be used in bioremediation of the soil

contaminated by pesticides and heavy metals in ecosystem. Cultivation of this

mushroom is an age-old practice.

Kapahi, M. &Sachdeva, S. (2017) stated that mushrooms, macro-fungi, are

among the nature’s most important mycoremediators. Pleurotus species (also called

oyster mushrooms) are considered to be the most popular and widely cultivated

varieties worldwide and this might be attributed to their low production cost and higher

yields. Apart from their nutritive and therapeutic properties, Pleurotus species have

high biosorption potential due to their extensive biomass, i.e. mycelial production.

On using oyster mushroom for mycoremediation

Oyster mushroom is the Pleurotus species, which is a well-known fungus that

can be used in bioremediation of the soil contaminated by pesticides and heavy

metals in ecosystem. Cultivation of this mushroom is an age-old practice. Though the

biological remediation properties were known from a very long time, as long as the

period of world war one, but only a little was done to commercialize it or incorporate it

in our daily lives. The absorption potential of Pleurotus species is still to be known to
the fullest extent.

On Using Oyster Mushroom for Mycoremediation

Oyster mushroom is the Pleurotus species, which is a wellknown fungus that

can be used in bioremediation of the soil contaminated by pesticides and heavy

metals in ecosystem. Cultivation of this mushroom is an age-old practice. Though the

biological remediation properties were known from a very long time, as long as the

period of world war one, but only a little was done to commercialize it or incorporate it

in our daily lives. The absorption potential of Pleurotus species is still to be known to

the fullest extent.

Mycoremediation is a method of clarifying heavy metals by fungal biomass,

using processes such as degradation, absorption, accumulation and conversion

through biological means. The microorganisms accumulate high amounts of heavy

metals inside them when grown in an area contaminated with these metals. oyster

mushroom breaks down a broad spectrum of organopollutants that are not easily

decomposed by any other biological agents (Nidhi Akkin, 2021). This ability makes

them promising organisms for use in various bioremediation projects. Thus, white rot

fungus are currently being used for soil remediation purposes utilizing techniques

such as land farming or composting technologies. They normally colonize plant

material (lignocellulosic), where their ability to degrade lignin, cellulose, and

hemicellulose makes them more antagonistic compared with other microorganisms.

(8) METHODOLOGY

The researcher will divide the experimentation into three key phases namely:
pre-experimental phase, experimental phase, and data collection and analysis phase.

PRE-EXPERIMENTAL PHASE

In this phase the researcher will prepare the model for the contaminated soils,

these are a. diesel contaminated soil, b. detergent contaminated soil, c. pesticide

contaminated soil, and d. used cooking oil contaminated soil.

A. Preparation of diesel contaminated soil

In the preparation of the diesel contaminated soil, the researcher will

follow the model used by Lopus&Bidoia (2009) in their study “Evaluation of

biodegradation of different types of lubricant oils in liquid medium.”

They utilized 0.15 ml chemical surfactant (Tween 80), 6.25 ml distilled

water per 100 g of soil. To verify the applicability of this, the researcher will

perform a confirmatory test. Where, the diesel contaminated soil will be

biodegraded for 60 days. After 60 days, seeds of pechay (Brassica rappa

L.) will be germinated to test its toxicity. The toxicity will be based on

the classification done by Lopez et.al (2010) in their study “Toxicity and

biodegradation in sandy soil contaminated of lubricant oils,” in which

according to them toxicity can be determined by growth inhibition. Above

40% inhibition the soil is toxic, between 10 – 40% inhibition is starting

toxicity, and below 10% inhibition the soil is non-toxic. Then, another 60

days of biodegradation and confirmatory testing of toxicity using the pechay

(Brassica rapa L.) seeds.

B. Preparation of detergent contaminated soil

In the preparation of detergent contaminated soil, the researcher will

 follow the model used by Mohamed et.al (2018) from their study “Effects of

detergents from laundry grey water on soil properties; a preliminary study.”

On which, they used ten (10) clothes and one (1) full cap of powder

detergent. Furthermore, the researcher will also utilize the water-detergent

ratio as suggested by Cameron (2007), which states that solutions of

detergents were made up a 0.5% w/v dilution. This was achieved by

dissolving 3.00 + 0.005 g of detergent in 600 ml of either deionized water

per 100 ppm hard water, which on this case the researcher will use well-

water.

Similarly, the soil will undergo biodegradation for sixty (60) days and

toxicity test using pechay (Brassica rapa).

C. Preparation of pesticide contaminated soil

In the preparation of pesticide contaminated soil, the researcher will

utilize Tamaran as pesticide to be used. Tamaran (methamidophos) was

the most prevalent type of pesticide used by the Benguet farmers. This type

of pesticide is an organophosphate pesticides cited in the study of Lu

(2010).

The researcher will apply the pesticide to the soil and similar to the

preparation of other soils its toxicity will be determined by its inhibition to

the growth of pechay seeds.

EXPERIMENTAL PHASE

A. Treatments

In this study three treatments will be utilized, treatment 1 is oyster

mushroom (Pleurotus ostreatus) mycelium, treatment 2 is paddy straw

mushroom (Volvariella volvacea) mycelium, and a control with no

treatment.

1. Spawn Preparation

In the preparation of spawn (f1), the researcher will follow the

standard protocol in mushroom production published by the

Department of Science and Technology – Industrial Technology

Development Institute (2014) entitled “Mushroom technology.”

a. PRODUCTION OF TROPICAL MUSHROOM (Straw

Mushroom)

A1. Preparation of Potato-Dextrose-Agar (PDA)

The following are the raw materials; fresh good quality

potatoes (200 g),dextrose powder (20 g), Agar bar (gulaman)

(20 g) and distilled water (1 L).

The procedure are as follows:

1. Wash, peel and dice the potatoes. Place 200 g in a

casserole where water has started to boil and allow to boil

until potatoes are soft enough for the palate.

2. Strain the broth (decoction) through cheesecloth.

Restore the volume of decoction to 1 L and put back into the

casserole.

3. Add the agar (chipped) and the dextrose powder. Heat

while stirring occasionally until the agar dissolves.

 4. Dispense 30 mL in each flat rhum bottle and plug the

mouth with the bottle cotton.

5. Sterilize the medium in a pressure cooker at 121°C or

15-lb. pressure for 15 minutes. Immediately after sterilization,

slant the test tubes at an angle of 20 to 25 degrees, making

sure that the agar does not touch the cotton plug.

6. Lay the bottles flat on the table until the agar congeals.

A2. Isolation of the Pure Culture (by Tissue Culture

Method) Tissue Culture Method (Volvariella volvaceae)

1. Select a good, young, healthy and fresh mushroom

(button stage for straw mushroom). Disinfect with 70%

rubbing alcohol using a cotton swab.

2. Cut vertically and horizontally half portion of the button

stage mushroom.

3. With a sterilized scalpel, cut approximately 1- cm cube

to the tissue between the cap and stem and place on the

middle of the plated agar.

4. Incubate for 5 to 7 days at ordinary temperature. This is

termed as pure tissue culture.

5. Transfer the pure culture into agar slants.

6. Incubate for 5 to 7 days at ordinary temperature. This is

now termed as sub-culture.

A3. Preparation of Spawn Substrates

 1. Place chopped dried substrate; i.e., rice straw, banana,

leguminous leaves in a suitable container and add water until

completely submerged. Place something heavy on top to

avoid floatation.

2. Ferment substrate anaerobically in water with urea (3

grams per gallon of water) as follows: chopped, dried tobacco

midribs - 3 days chopped, dried kakawati leaves - 5 days

chopped, dried ipil-ipil leaves - 5 days chopped, dried rice

straw - 3 days chopped, dried water lily - 2 days chopped,

dried banana leaves - 3 days

3. Wash the substrate with tap water three times or until

objectionable odor is removed.

4. Mix with sawdust at a proportion of two parts substrate

to one (2:1) part sawdust.

5. Add rice bran (Class A) at 20% of the major substrate.

6. Readjust the moisture at 65% to 70% (damp moisture).

7. Place substrates in polypropylene bags (PP) and 500

g/bag. Use 6x10 PP bags and pull-end of the bag, pass thru a

PVC pipe ring (1” long x 1” dia.) Plug with used cotton, cover

with scratch paper and tie with a rubber band.

8. Sterilize at 15-lb. pressure for 1 to 1½ hours or steam

for 4 hours in a drum.

9. Cool, inoculate with pure culture. Inoculation of the

 Spawn 1. Sterilize the inoculating needle in the flame of an

alcohol lamp. 2. Lift from the inoculums about 1.5 cm² and

transfer into the bagged substrate.

3. Flame the lip of the bag as well as the lip of the rhum

bottle containing the inoculums before lifting a portion for

transfer.

4. The inoculums substrate is now termed spawn and is

ready for planting into beds after two weeks.

b. PRODUCTION OF SEMI-TROPICAL MUSHROOM (Oyster

Mushroom) Preparation of Potato-Dextrose -Agar (PDA)

Follow the preparation of PDA as discussed in the

production of tropical mushroom.

B1. Preparation of Substrates (Fruiting bags)

1. Mix the following materials thoroughly: Sawdust, dried

sieve - 78% Rice bran, class A - 20% Calcium carbonate - 1%

Refined sugar - 1% 100%

2. Add tap water sparingly until the mixture reaches

approximately 65-70% moisture. When a handful of the mixture is

pressed in the palm of the hand and no water runs off in between

the fingers and will stay in form after the release of pressure, the

65-70% moisture is reached.

3. Pile and pack the substrate in pyramidal form.

4. Cover with plastic sheet and incubate for 5 days. Re-

pile on the 3rd day.

5. On the fifth day, aerate the piled material by spreading

the material thinly in a shaded area to remove the toxic gases

produced during the fermentation period. Note : Do not spread

the material under the sun.

6. Pack 1 kg of the material in PP bags after 2-3 hours

aeration. The smell of the toxic gas is removed and its moisture

is re-adjusted to 65-70% level.

7. Collect the upper part of the plastic bag and pass it thru

PVC pipe ring (1” dia. x 1” length), then pull the plastic thru this

pipe. Hold the free end of the plastic bag with a rubber band. 8.

Plug the bag with cotton and provide with paper to lessen

moisture uptake during the sterilization process.

9. Sterilize the packed mixture at 15-lb pressure for 1 – 1½

hours.

10. Cool the substrate.

This is now ready for inoculation with Pl sp mold grown on

PDA or sorghum grains.

B2. Propagation of the Mushroom Mold

1. Inoculate the PDA with young and pure culture of the

Pleurotus mold.

2. Incubate the pure culture at 25°C for two weeks or until

the full ramification of mycelium is observed. (This is now termed

as subculture.) This is now ready for the inoculation of mother

spawn.

B3. Preparation of the Mother Spawn

1. Wash the sorghum grains thoroughly under running tap

water.

2. Place in a casserole, cover with water at about 2” above

grain level.

3. Bring to a boil with occasional stirring to test the grains.

4. Stop boiling when grains are just about to burst

(malabo/maligat stage).

5. Immediately strain water to prevent grains from

becoming overcooked. Use fine screen or cheesecloth for the

purpose.

6. Cook briskly. When grains are merely damp, distribute

in empty flat rhum bottles. One kilo will make 10 bottles.

7. Plug bottles with absorbent cotton, support with a piece

of paper and rubber band. Sterilize at 15 psi for 15 minutes. Then

cool.

8. Inoculate with 15-day old pure culture of the mold.

(parent-tissue culture).

9. Incubate at 28-30°C until the whole medium is fully

impregnated with the mushroom mold (normally 10-15 days).

These are now the mother spawns, one of which will be good for
 25 to 30 spawn bags.

B. Application of treatments to contaminated soils

The researcher will use 20 kg of each contaminated soil to replicate the study

conducted by Stamets (2005) in his study about “Oil spill and mycoremediation.” Each

contaminated soil will be replicated three times and will be randomly assigned by the

treatments.

Treatment 1 Treatment 2 Treatment 3

S1 S1 S1

Block 1 S1 S1 S1

S1 S1 S1

S2 S2 S2

Block 2 S2 S2 S2

S2 S2 S2

Block 3 S3 S3 S3

S3 S3 S3

S3 S3 S3
 S4 S4 S4

Block 4 S4 S4 S4

S4 S4 S4

Table: Experiment design layout of the study using complete block design

Block 1 is for the diesel contaminated soil, block 2 is for detergent

contaminated soil, block 3 is for pesticide contaminated soil, and lastly block 4 is for

used-cooking oil contaminated soil.

B1. Application of the spawn to the contaminated soils

The developed spawn with its substrate (base material) will be buried in

the contaminated soil the same way in the study of Reddy and Matthew in their study

“Bioremediation with white rot fungi,” it will be shaded 65%, allowing it to receive

natural precipitation.

C. Data collection and analysis phase.

To assess the mycoremediation potential of the treatments, the following

parameters will be explored. First is the mushroom growth, second is the growth

inhibition of the soil as indication of its toxicity, and lastly the soil quality.

C1. Mushroom Growth

 Mushroom growth will be recorded after 2 weeks and 5 weeks for each

replicate of soil contaminated soils.

C2. Plant germination

Planting of fifty (50) pechay (Brassica rapa L.) seeds will be performed

after 9 weeks to each of the contaminated soil to test its toxicity by utilizing its growth

inhibition potentials.

C3. Soil Quality

The soil samples will be analyzed to test the quality of the soil using

basic soil parameters.

D. Statistical Analysis

Each block will be treated separately and as a whole, to determine the

significant difference of the treatments.

Each block will be treated with one-way ANOVA based on the three different

parameters mentioned. And then, two-way ANOVA will be employed to see if there

are significant mean difference among the blocks based on the data collected from

the three parameters mentioned.

(9) EXPECTED OUTPUTS AND POTENTIAL IMPACTS

Fungi, such as mushrooms, can reduce even the most complex aromatic

hydrocarbons—the primary component of oil—because of their specific enzymes. In

this study, two species of mushroom, oyster mushroom (Pleorotus ostreatus) and

paddy straw mushroom (Volvariella volvacea), will be produced through traditional

spawning to be utilized as treatments for oil-contaminated piles of soil. Four different

contaminants, namely diesel, detergent, pesticide, and used cooking oil will be used

to four piles of soil. Each contaminated soil will be replicated three (3) times using

20kg each and be randomly treated or left untreated. Observation will be conducted

for sixty (60) days; subsequently, 50 pechay (Brassica rapa) seeds will be planted on

each contaminated soil to test its toxicity.

This research will result in a cohesive and wide overview of oil contaminated

soil response to paddy straw mushroom (Volvariella volvacea) across the soil base of

the local town of Nabua, Camarines Sur, as well as an exposition to effectively grow

mushroom for potential oil-contaminated soil degradation and identify the indications

of soil toxicity due to oil spillage.

These outputs are of paramount importance for understanding

 Potential mycoremediation effects of mushroom species

 Different types of oil responses to mushroom species

 The harm and signs of soil toxicity, and

 Effective treatment for oil spillage

Concrete outputs will include a comparative assessment mainly between

(Pleorotus ostreatus) and paddy straw (Volvariella volvacea) in mycoremediation of

soil in Nabua, Camarines Sur contaminated with four different contaminants.

The present research considers the effects of oil spillage to soil toxicity and growth

inhibition. In this sense, it will make a significant contribution to the geotechnology,

agriculture, and biodiversity in the Philippines. This will be most essentially significant

to farmers and foresters as it will provide them a toxic-free soil for the growth of their

products. In return, vendors will be provided with high-quality products grown from a

non-toxic environment. This shall also benefit the consumers who aim for healthy

eating. Industrialists, environmentalists, and promoters of green spaces who wish to

address on-site pollution are also stakeholders as this will be the way to further

promote their cause. Contaminated land experts will also be provided with a wide

scope in the treatment of contaminated land. Community members will also benefit as

this will provide them a pollution-free environment.

(10) WORKPLAN AND TARGET

Objectives Tasks/ Timeline Person/ Resource Budgetary

Activity People s Requirement

Responsibl

To review The researcher 2022 Researcher Potatoes 2,000- 5,000

the potential should first pesos

of paddy prepare the Dextrose

straw production of powder

mushroom Volvariella

(Volvariella volvacea. First is Agar bar

volvacea) to prepare the (gulaman)

for oil- Potato-Dextrose-

contaminate Agar (PDA), next Distilled

d soil is isolation of water

degradation the Pure Culture

(by Tissue Chemical

Culture Method) surfactant

Tissue Culture

Method Distilled

(Volvariella water

volvaceae), then

Preparation of Crates or

Spawn containers

Substrates. For

the preparation of Beakers

oil-contaminated

soil the

researcher will

follow the model

used by

Lopus&Bidoia. in

their study

“Evaluation of

biodegradation of

different types of

lubricant oils in
 liquid medium.”

Utilize 0.15 ml

chemical

surfactant

(Tween 80), 6.25

ml distilled water

per 100 g of soil.

the soil will

undergo

biodegradation

for sixty (60) days

and toxicity test

using pechay

(Brassica

rapa).Prepare the

treatment and

materials needed

to conduct the

experiment, then

record the

outcome.

To The researcher 2022 Researcher Indicator

determine will observe the for soil

the significant quality

mushroom differences of

growth, each treatment. Pechay

growth They can use the seeds

inhibition of gathered data as

the soil as basis on T-square

an determining ruler

indication of themushroom

its toxicity, growth, growth Crates or

and soil inhibition of the containers

quality of soil as an

the blocks indication of its

using the toxicity, and soil

different quality of the

treatments blocks using the

different

treatments.

To Observe the 2022 Researcher Crates or

determine three different containers

the oil treatments on

degradation each Grown

ability of the contaminated soil Pechay or

three by visual Adult

 treatments inspection and Pechay

write or collect

the gathered

information about

the different

results on each

treatment.

To find out if Make use of one- 2022 Researcher Gathered

there is a way data

significant ANOVA to Research

difference analyze the teacher

between the gathered data

different

treatments

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