Abstract

The study entitled “Fungal Isolates from Oil Contaminated Soil for Oil Spill

Degradation” aims to determine the mean of Zone of Clearing of Fungal Isolates A and B

when treated on oil spill and to determine if there is a significant difference in the Zone of

Clearing (ZOC) of oil spill when treated with fungal isolates A and B. Isolates A and B from

oil contaminated soil will be isolated and incubated in Potato Dextrose Agar. Isolates A and

B with largest colonies will be subjected to Butterfat Assay in Butterfat to determine the zone

of clearing. The experiment found out that in fungal isolate A, isolate 2 has the highest mean

of zone of clearing with a mean of 23.3333333. Isolate 4 has the lowest mean of zone of

clearing with the value of 8.66666667. Isolate 1 has a mean of 16.3333333, Isolate 3 has a

mean 17.6666667 and isolate 5 with a mean of 14. In fungal isolates B, the results shows that

isolate 10 has the highest mean of zone of clearing with a mean of 25.3333333. Isolate 7 has

the lowest mean of zone of clearing with the value of 15. Isolate 8 has a mean of 21.6666667;

Isolate 6 has a mean 18.3333333and Isolate 5 has a mean of 18. 3.The F-test ANOVA shows

that Sig<0.05. Therefore, there is no significant difference between the zone of clearing of oil

spill when treated with Fungal Isolates A and B The following recommendation of this study

are to determine the turbidity of the oil spill when treating using fungal isolates, determine

the characteristics of the fungal isolates used in oil degradation and test the amount of lipase

produced by the fungal isolates.

Keywords: Zone of Clearing, Butterfat Assay, Oil Spill degradation.

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

CHAPTER 1

INRODUCTION

1.1 Background of the study

Petroleum based products doesn’t just fuel everyday living but mostly

industrialization in global prospect. Petroleum products powers electricity generators, propels

vehicles and fuels companies for making chemicals, plastics or any material that is included

in everyday use. According to U.S. Energy Information Administration, the world consumes

more than 85.5 million barrels of oil each day. Each barrel holds about 42 gallons of oil. So

the world is consuming 3.6 billion gallons of oil every day.

Although found to be helpful at many aspects, petroleum oil also contributes to

environmental concerns and issues. About 53% of petroleum leaks and spills during its

extraction, refining, and industrialization (Kvenvolden, K. A. and Cooper C., 2003).

Common problem encountered with the use of petroleum products is oil spills.

Concerning every person in the society, oil spills affects economic, social and environmental

facet. Moreover, the chemicals to be used in breaking up the oil can be toxic making it more

difficult to eliminate the spilled oil. Accordingly, it can still take up to a decade or more for

an area to recover from the said issue.

The clean-up and remediation of an oil spill is not easy. There are variables that are

needed to be considered such as type of oil, location of the spill, amount of oil spilled,

weather conditions, and proximity to delicate areas are always different. Thus clean-up

2
 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

professionals must be prepared to face a variety of problems both known and unknown

(Dardis, D. and Pendarvis, P., 2010).

Several methods using various technologies have been developed through the years to

cleanup oil spills. In some regions dispersants are employed to speed up the natural process

of the breakdown of oil. Unfortunately many dispersants are dangerous to wildlife. Recently

some researchers have genetically engineered bacteria to breakdown oil. Many

microorganisms can serve to break oil down into harmless substances such as fatty acids and

carbon dioxide. This action is known as biodegradation.

In the present study, sand samples contaminated with oil spill were collected from

Pensacola beach (Gulf of Mexico) and tested to isolates fungal diversity associated with

beach sands and investigates the ability of isolated fungi for crude oil biodegradation. From

sixteen fungal strains, four strains were confirmed for biodegradation ability of crude oil, the

isolated fungi belongs to Aspergillusniger with higher activity followed by

Penicilliumdocumbens , Cochlioboluslutanus and Fusariumsolani . Aspergillusniger recorded

the highest weight loss of 8.6%, Penicilliumdocumbens (7.9 %) and Cochlioboluslutanus

(4.7%) whereas the lowest weight loss was demonstrated by Fusariumsolani strain 421502

(1.9%) (Al-Nasrawi, H., 2012).

In this science investigatory project, the researchers would like to isolate, produce and

characterize lypolitic fungi from oil contaminated soil for degradation of petroleum oil.

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

1.2 Research Question

The study entitled “Fungal Isolates from Oil Contaminated Soil for Oil Spill

Degradation” generally aims to determine if fungal isolates from oil contaminated soil are

effective for degradation of oil.

Specifically it aims to answer the following questions;

1. What is the mean of Zone of Clearing (ZOC) of oil spill when treated with fungal isolates A

and B?

2. Is there a significant difference in the Zone of Clearing (ZOC) of oil spill when

treated with fungal isolates A and B?

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

1.3 Research Objectives

The study entitled “Fungal Isolates from Oil Contaminated Soil for Oil Spill

Degradation” generally aims to determine if fungal isolates from oil contaminated soil can

degrade oil.

Specifically it aims to:

1. Determine the mean of Zone of Clearing (ZOC) of oil spill when treated with fungal isolates

A and B.

2. Determine if there a significant difference in the Zone of Clearing (ZOC) of oil spill

when treated with fungal isolates A and B.

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

1.4 Research Hypothesis

These are the following hypotheses that were formulated based on the problem

statement.

Null Hypothesis (H0)

1. There is no significant difference in the Zone of Clearing (ZOC) of oil spill when

treated with fungal isolates A and B.

Alternative Hypothesis (H1)

1. There is significant difference in the Zone of Clearing (ZOC) of oil spill when treated

with fungal isolates A and B.

1.5 Conceptual Framework

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 Fungal Isolate Clearing
A and B on Subject to (ZOC) of
Oil Butterfat
Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

Contaminated Assay Fungal

Soil Isolates A
Figure 1: The Schematic diagram of Isolation of Fungi for oil degradation

and B
Figure 1 shows that fungal isolates A and B from oil contaminated soil from

Barangay 2, San Francisco, Agusan del Sur, Philippines was subjected to butterfat assay to

determine the Zone of Clearing formed by Isolates A and B.

1.6 Scope and Limitation

This study focus in isolating, producing, and optimizing the fungi isolated from the

oil contaminated soil to test its capability to degrade oil spills. Oil contaminated soil from

Barangay 2, San Francisco, Agusan del Sur are used as the source of the fungi for

biodegradation of oil spills.

This study is limited only to determine the Zone of Clearing (ZOC) formed in

butterfat by top 5 fungal isolates A and B with highest number of colonies and determining

the turbidity of the oil spill, characterization of the fungal isolates and amount of lipase

produce by the isolates are not included in the study.

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

1.7 Significance of the study

Oil spills is one of the most noticeable forms of damage to the marine environment.

Oil enters the seas not only as a result of spectacular oil tanker or oil rig disasters, but also –

and primarily – from diffuse sources, such as leaks during oil extraction, illegal tank-cleaning

operations at sea, or discharges into the rivers which are then carried into the sea (World

Ocean Review, 2010).

Commonly, some countries prefer to use oil skimmers or booms that acts as floating

barriers, while others involve use of dispersants. The effectiveness of these chemicals is

heavily dependent on the type and condition of the oil, however. A further limiting factor is

that these dispersants can generally only be used for a short time after the spill has occurred,

8
 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

as the chemical and physical processes described above begin to impair their effectiveness

after only a few hours (World Ocean Review, 2010).

Biodegradation is also advantageous due to its environmentally friendly approach. It

allows for natural organisms to degrade the toxic hydrocarbons into simple compounds

which pose no threat to the environment, and this also eliminates the need to remove and

transport the toxic compounds to another site (Radermacher, M., 2009).

This study can add up to help answer the problem of marine oil spill through

environment friendly proposed solution by finding potential oil spill degrading fungus from

oil contaminated soil.

1.8 Definition of Key Terms

Biodegradation: It is the method of degrading of oil spill by using fungal isolates

Isolation: Isolation is the process of extracting the fungi from the oil contaminated soil.

Zone of Clearing: It refers to the halos formed by the fungal isolates in the butterfat.

Butterfat Assay- It is the method used to determine the zone of clearing formed by fungi

isolates in butterfat.

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

1.9 Review of Related Literature

This chapter includes ideas, finished thesis, generalization or conclusions,

methodologies and others. Those that were included in this chapter helps in familiarizing

information that are relevant and similar to the present study.

Extracellular enzymatic activity profiles in fungi isolated from oil-rich environments

About 34 wild fungal species associated with edible oil mill wastes were isolated by

the serial dilution technique. Methods for rapid screening of fungal species against

production of extracellular enzymes such as amylase, protease, cellulase, and lipase are

reported. Among all the species, Aspergillusversicolor exhibited high amlylolytic and

10
 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

gelatinolytic activity, whereas Penicilliumcitrinum showed only high amylolytic activity.

Maximum cellulolytic activity was recorded for Absidiacorymbifera, As. niger,

Cunninghamellaechinulata, Curvularialunata, Fusariumsolani, Mucorracemosus,

Paecilomycesvariotii, and Syncephalastrumracemosum.The fungal species Ab. corymbifera,

As. fumigatus, As. japonicus, As. nidulans, As. terreus, Cun.verticillata, Cur. pallescens, F.

oxysporum, Geotrichumcandidum, M. racemosus, Pe. citrinum, Pe. frequentans,

Rhizopusstolonifer, andTrichodermaviride exhibited maximum lipase activity. This study

confirms the isolated fungi present on a wide range of substrates in the ambient environment,

and these results could provide basic data for further investigations on fungal extracellular

enzymes.

Lipolytic activity of fungi on rapeseed oil

Many species of fungi grow on stored seeds of oilseed rape causing the development

of high levels of free fatty acids. However, the presence of a fungus on the seed does not

imply that it is lipolytic. The work reported here was performed to test the ability of fungi

isolated from stored rapeseed to release fatty acids from refined rapeseed oil. Lipolytic

activity was assessed by growing fungi on agar, incorporating the oil as droplets and the

indicator Nile blue sulphate. Of 18 species tested, only Aspergillusversicolor showed no

lipolytic activity. Another method of testing for lipolysis was used for three of the fungi.

They were grown in a liquid medium containing oil and the amount of free fatty acids was

11
 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

measured titrimetrically. The results show that many fungi can cause lipolysis of rapeseed

oil, but there are great differences in the lipolytic activity between species.

Crude oil degradation by microorganisms isolated from the marine environment

The utilization of crude oil by microorganisms isolated from marine environments

was investigated. Enrichment procedures for isolating crude oil degrading organisms were

carried out using samples of mud collected among the estuaries and along the coast of North

Carolina. The basal medium was seawater supplemented with nitrogen and phosphate. The

fungi isolated under these conditions utilized crude oil more effectively than did the bacteria

and several oil utilizing fungi were isolated in pure culture. Among the fungi selected were

strains of Cunninghamellaetegans, Aspergillusversicolor, Cephalosporiumacremonium, and a

Penicillium sp. These fungi utilized a wide variety of hydrocarbon substrates as a source of

carbon and energy. All grew on a mineral salts medium with no requirement for seawater.

The amount of crude oil utilized by growing fungi was determined. The results

suggested that of the fungi isolated C. elegans and the Penicillum sp. were most effective in

degrading crude oil. When paraffin base crude oil was added to the seawater basal salts

medium (0.2% v/v) and inoculated with C. elegans over 90% of the oil was assimilated after

5 days growth. Less than one‐half of this amount of asphalt base crude oil was utilized. The

optimum temperature for growth of fungi that utilized crude oil differed but all grew at

temperatures between 15 and 24 °C. Significant growth of the fungi on crude oil did not

occur unless the seawater was supplemented with a source of nitrogen and phosphorus. The

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

results of this study suggested that fungi can effectively assimilate crude oil and that paraffin

base crude is more readily degraded than the asphalt base crude oil.

Biodegradation of aliphatic hydrocarbon by indigenous fungi isolated from used motor

oil contaminated sites

Eighteen indigenous fungal isolates has been successfully isolated from samples of

used motor oil, top five centimetres of soil and drainage water contaminated with used motor

oil. All of the pure fungal isolates obtained were identified, characterized and subjected to

preliminary screening by evaluating the average growth rate of each fungal isolates on

minimal media containing 1% (v/v) used motor oil. Trichodermaasperellum strain TUB F-

1067 (SA4), Trichodermaasperellum strain Tr48 (SA5), Trichodermaasperellum strain TUB

F-756 (SA6), Penicillium species (P1), and Aspergillus species (P9) were further selected for

their hydrocarbon biodegradation potential. Among these five fungal isolates selected, P1

strain presented a significant degree of degradation by degrading almost all of the n-alkanes

(n-C-15 to n-C-23 range) present in the used motor oil, thus of greater potential in degrading

the aliphatic hydrocarbon compounds of used motor oil.

Crude oil and hydrocarbon-degrading strains of Rhodococcusrhodochrous isolated

from soil and marine environments in Kuwait

Soil and marine samples collected from different localities in Kuwait were screened

for microorganisms capable of oil degradation. Both fungi and bacteria were isolated. The

fungal flora consisted of Aspergillusterreus, A. sulphureus, Mucorglobosus, Fusarium sp. and

Penicillumcitrinum. Mucorglobosus was the most active oil degrading fungus isolated.

Bacterial isolates included Bacillus spp. Enterobacteriaceae, Pseudomonas spp., Nocardia

13
 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

spp., Streptomyces spp.,andRhodococcus spp. Among these Rhodococcus strains were the

most efficient in oil degradation and, relatively speaking, the most abundant. Bacterial and

fungal isolates differed in their ability to degrade crude oil, with Rhodococcus isolates being

more active thatfungin in n-alkane biodegradation, particularly in the case of R. rhodochrous.

In addition to medium chain n-alkanes, fungi utilized one or more of the aromatic

hydrocarbons studied, while bacteria failed to do so. R. rhodochorous KUCC 8801 was

shown by GLC and post-growth studies to be more efficient in oil degradation than isolates

known to be active oil degraders.

White rot fungi and their role in remediating oil-contaminated soil

White rot fungi, which utilize lignin as an energy source, possess the ability to

degrade a wide spectrum of environmental pollutants using peroxidases enzymes. This ability

led to several studies that focused on the development of bio-treatment systems using white

rot fungi. Three strains of white rot fungi, namely Phanerochaetechrysosporium,

Pleurotusostreatus, and Coriolusversicolor, have been tested for their ability to degrade oil in

contaminated soil. A soil microcosm test was designed to study the effect of strain used,

inocula concentration, and the addition of nitrogen on bioremediation efficiency.

Coriolusversicolor showed the highest degradation rate, as the total petroleum hydrocarbon

(TPH) concentration decreased from 32 g/kg to reach 7 g/kg within 12 months. The increase

in the inocula concentration enhanced substantially TPH degradation. The nutrient rich

conditions initiated the growth of other soil existing microorganisms enabling them to

degrade different compounds synergistically with some white rot fungal strains. Because of

their unique biodegradative abilities, white rot fungi are considered potentially useful

microorganisms for bioremediation applications.

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

Oil-degrading capabilities of yeasts and fungi isolated from coastal marine

environments

Seventy-four yeasts and 224 fungi were isolated from marine water and sediment

samples taken from the Strait of Juan de Fuca and northern Puget Sound. When these isolates

were grown in the presence of Prudhoe Bay crude oil, only three yeasts and 63 fungi were

able to degrade some or all of the n-alkanes. None degraded the isoprenoids, pristane and

phytane. Forty-seven isolates were identified as Penicillium species and of these, 39 attacked

the n-alkanes in the crude oil. Twelve organisms which degrade n-alkanes were tested for

their ability to mineralize [14C]naphthalene and [14C]phenanthrene which had been added to

the crude oil. No 14CO2 was detected from any of the cultures containing these compounds.

Capillary gas chromatographic analyses of the aromatic fractions from these 12 cultures

showed no loss of hydrocarbons or sulfur hetero-cycles, indicating that they were unable to

completely or partially oxidize any of the resolvable compounds in this fraction.

Changes in mutagenicity during crude oil degradation by fungi

Two fungal strains, Cunninghamellaelegans and Penicilliumzonatum, that grow with

crude oil as a sole carbon source were exposed to three crude oils that exhibit a range of

mutagenic activity. At regular time intervals following fungal incubation with the various

crude oils, extracts were tested for the presence of mutagenic activity using the spiral

Salmonella assay. When the most mutagenic of the oils, Pennsylvania crude oil, was

degraded by C. elegans or by P. zonatum, its mutagenicity was significantly reduced;

corresponding uninoculated (weathered) controls of Pennsylvania crude remained mutagenic.

15
 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

West Texas Sour crude oil, a moderately mutagenic oil, exhibited little change in

mutagenicity when incubated with either C. elegans or P. zonatum. Swanson River Field

crude oil from Cook Inlet, Alaska is a slightly mutagenic oil that became more mutagenic

when incubated with C. elegans; weathered controls of this oil showed little change in

mutagenicity. Mycelial mat weights measured during growth on crude oils increased

corresponding to the biodegradation of about 25% of the crude oil.

Role of Pycnoporuscoccineuslaccase in the degradation of aromatic compounds in olive

oil mill wastewater

In a previous work was reported the ability of Pycnoporuscoccineus to decolorize

olive oil mill wastewaters (OOMW) without an additional carbon source. We studied the

composition of the enzymatic system involved in the process. The fungus secreted only

laccase under the different culture conditions studied even in presence of compounds

promoting the production of peroxidases. The highest laccase levels were attained in Cu2+–

ethanol medium (100,000 U/l after 45 incubation-days). A single isoenzyme was purified

with a yield of 79%. This laccase is a glycoprotein (8% N-linked carbohydrate) with a

molecular mass and pI of 61.5 kDa and 3.7, respectively. The highest oxidation rate was

obtained around pH 3.5 for ABTS and DMP and the highest DMP oxidation at 60 °C. The

enzyme was stable at pH 7 at room temperature and showed a half-life of 8 and 2 h at 50 and

60 °C, respectively. The treatment of OOMW with the laccase showed similar results to

those reported with the fungus indicating that laccase plays an important role in the

degradative process. The high levels of laccase secreted by P. coccineus and its stability

suggest that it could be a useful tool for this and other environmental applications.

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

Hydrocarbon-degrading filamentous fungi isolated from flare pit soils in northern and

western Canada

Sixty-four species of filamentous fungi from five flare pits in northern and western

Canada were tested for their ability to degrade crude oil using gas chromatographic analysis

of residual hydrocarbons following incubation. Nine isolates were tested further using

radiorespirometry to determine the extent of mineralization of model radiolabelled aliphatic

and aromatic hydrocarbons dissolved in crude oil. Hydrocarbon biodegradation capability

was observed in species representing six orders of the Ascomycota. Gas chromatography

indicated that species capable of hydrocarbon degradation attacked compounds within the

aliphatic fraction of crude oil, n-C12 - n-C26; degradation of compounds within the aromatic

fraction was not observed. Radiorespirometry, using n-[1-14C]hexadecane and [9-

14C]phenanthrene, confirmed the gas chromatographic results and verified that aliphatic

compounds were being

mineralized, not simply transformed to intermediate metabolites. This study shows that

filamentous fungi may play an integral role in the in situ biodegradation of aliphatic

pollutants in flare pit soils.

Fungi and Bacteria Isolated from Two Highly Polluted Soils for Hydrocarbon

Degradation

Cultivable fungi and bacteria were isolated from two highly contaminated soils with

total petroleum hydrocarbon (TPH) concentrations of 60,600 and 500,000 mg kg–1. The aim

of the study was to determine the capacity of these bacteria and fungi to degrade TPH,

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

specifically the aliphatic hydrocarbon (AH) and polycyclic aromatic hydrocarbon (PAH)

fractions, when cultivated in a mineral liquid culture (modified Rennie medium) together

with a complex mixture of TPH as the unique source of carbon. Thirty-seven hydrocarbon-

degrading strains were isolated, but only six strains showed a high ability to degrade PAHs,

AHs and TPH. These strains were identified as Pseudomonas pseudoalcaligenes, Bacillus

firmus, Bacillus alvei, Penicilliumfuniculosum, Aspergillussydowii and Rhizopussp., and

they removed 79%, 80%, 68%, 86%, 81% and 67% of TPH, respectively. P.

pseudoalcaligenes and P. funiculosum removed 75% of PAHs, while B. firmus and P.

funiculosum removed 90% and 92% of AHs, respectively. The highest TPH removal was

observed by P. funiculosum, which was isolated from the soil with a high TPH concentration.

A. sydowii was also isolated from this soil; no reports were found regarding its capacity to

remove PAHs, but it was able to degrade five- and six-ring aromatic compounds.

Studies on relative capabilities of bacterial and yeast isolates from tropical soil in

degrading crude oil

The relative capabilities of two bacterial isolates Serratiamarcescens OCS-21 and

Acinetobactercalcoaceticus COU-27 from Ebubu oil polluted soil of Rivers State, Nigeria

and a yeast isolate, Candida tropicalis PFS-95 from unpolluted soil of the University of

Calabar campus, in degrading transniger pipeline crude oil were investigated. Crude oil

degradation was measured by gravimetric analysis and capillary gas chromatographic

techniques. Candida tropicalis PFS-95 exhibited a more efficient ability in degrading the

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

crude oil than either S. marcescens OCS-21 or A. calcoaceticus COU-27. The yeast isolate

PFS-95 degraded 68.9% of crude oil after 16 days of incubation whereas OCS-21 and COU-

27 degraded 51.5% and 45.5% of crude oil respectively over the same incubation period.

Capillary gas chromatographic analysis of the degraded crude oil revealed that crude oil

components of chain length C12 to C32 were extensively degraded by PFS-95 after 16 days

of incubation while OCS-21 caused a moderate degradation of the crude oil components

(C20 to C28) over the same period. Acinetobactercalcoaceticus COU-27 was able to degrade

only C22 to C30 components of the crude oil after 16 days of incubation. Results of crude oil

adherence/emulsification tests revealed that OCS-21 was unable to emulsify the crude oil but

exhibited maximal adherence to the crude oil whereas PFS-95 and COU-27 exhibited

minimal adherence to, but maximal emulsification of the crude oil. Results obtained suggest

that C. tropicalis PFS-95 may be a better microorganism for clearing oil spills in tropical

soils than either S. marcescens OCS-21 or A. calcoaceticus COU-27.

Degradation of the Lauric acid oils

The composition, properties, uses and degradation of the lauric acid oils have been

discussed. Coconut and palm kernel oil contain approximately 40% medium chain fatty acids

(C6:0–C12:0). The medium chain fatty acids in these oils are readily degraded by

filamentous fungi belonging to the following genera, Penicillium, Aspergillus,

Cladosporium, Fusarium, Trichderma and Monascus to give the methyl ketone one carbon

atom less. This type of spoilage has been described as ketonic rancidity. Ketonic rancidity

caused by Penicilliumcrustosum can be controlled by the total absence of fungal spores, a

19
 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

temperature less than 4°C, water activity less than 0·91; the addition of sorbic acid at 1000

mg/kg or by the absence of oxygen. Coconut oil is a better substrate for ketonic rancidity

than palm kernel oil due to the higher concentration of medium chain fatty acids in the

former.

CHAPTER 2

METHODOLOGY

This section presents a discussion of method, design, and layout, the research

variables, the subjects, and sampling procedures, the research tools and instruments, and the

statistical procedures utilized for the analysis and interpretation of the data.

2.1 List of Materials

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

The following materials and media used in this study: Oil Contaminated Soil, 100

mL graduated cylinder, distilled water, ruler, flask, toothpick, petri dish, Potato dextrose

agar, distilled water; incubator, gloves, masks, disposable pipette, cylinder, cotton, zip lock

bags, felt tip pen, Potato dextrose broth, butterfat, inoculation loop, streptomycin, alcohol

lamp, ethanol.

2.2 Research Design

Table 1: Zone of Clearing of Top Five Black Fungi Isolates and Top Five White Fungi

Isolates.

Test
Zone of Clearing(Fungal Isolate A) Zone of Clearing(Fungal Isolate B)
Independent
R1 R2 R3 Average R1 R2 R3 Average
Variable
Isolate #1 X1 X2 X3 X4 Y1 Y2 Y3 Y4
Isolate #2 X1 X2 X3 X4 Y1 Y2 Y3 Y4
Isolate #3 X1 X2 X3 X4 Y1 Y2 Y3 Y4
Isolate #4 X1 X2 X3 X4 Y1 Y2 Y3 Y4
Isolate #5 X1 X2 X3 X4 Y1 Y2 Y3 Y4

Legend: X- Zone of Clearing (Halos) of Isolate A isolates in millimeters

Y- Zone of Clearing (Halos) of Isolate B isolates in millimeters

Isolates A and B from oil contaminated soil was isolated and incubated in Potato

Dextrose Agar. Isolates A and B with largest colonies is subject to Butterfat Assay in

Butterfat to determine the zone of clearing.

2.3 Procedures

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

1. Collection of Sample

Fifty grams of Oil contaminated soil was collected

at Chito’s Machine Shop, Brgy. 4, San Francisco,

Agusandel Sur. The collected soil was placed in a

clean zip lock bags and containers to protect from

contamination.

Collection of Sample

2. Preparation of Medium

The medium that is used for culturing the fungi is prepared using potato

dextrose agar and distilled water. 500 mL of

distilled water and 12g grams of potato dextrose

agar is mixed in the Erlenmeyer flask then the

PDA is dispensed equally in petri dishes. To make

sure that only fungi will grow on the plates, each

plate is added 0.1 mL of chloramphenicol or

streptomycin.

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

Preparation of PDA

3. Isolation of fungi from the Oil Contaminated Soil

Using serial dilation,10 grams of soil

sample is diluted using 90mL distilled water and

shake it (10-1) then 10 mL from the (10-1) is

transferred to 90mL distilled water (10-2), as well

as for (10-3) and (10-4). After the dilution, the

solution is plated in the potato dextrose agar

medium using sterile L-rod and is incubated for

24 hours.

Dilution of Oil Contaminated Soil

4. Purification of the fungi from the Oil Contaminated Soil

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

After incubation, streaking of the fungi isolates then

followed by purification of fungi isolates is done on

separate plates and then the plates is divided in

quadrants. Then the isolates are incubated for 72

hours.

Purification of Isolates by Streaking

5. Screening of the Isolates

A medium are used (Butterfat Agar, 5mL

butterfat, 10mL Potato Dextrose Agar). Four

hundred mL of Potato dextrose Agar and 200

mL of Butterfat are prepared. The butterfat are

extracted by heating on a hotplate and are

separated from its curd by decantation and

filtration processes. After that, it is dispensed on

a 1000 mL Erlenmeyer flask and sealed and

sterilized using an autoclave. The butter fat and

Recording the Zone of Clearing potato dextrose agar are mixed and each plate

(ZOC) have a ratio of 5:10, then it is transferred in

three (3) 500 mL Erlenmeyer flask and shaken

for five (5) minutes. After that, it is dispensed to

24
 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

40 clean and sterile petri dish containing 15 mL

of the medium each and is dried. After drying

the medium in the plates, the fungal isolates is

inoculated in quadrant using sterile toothpick,

place in a clean zip-lock bags and incubate for

72 hours. After incubation, the zone of clearing

is recorded using ruler.

6. Disposal of Waste

A l l t h e a p p a r a

contaminants in the apparatus. All the used materials

is properly washed and stored in each respective

container. Then all disposables are disposed in their

corresponding garbage bins.

Materials used are purified using autoclave

CHAPTER 3

RESULTS AND DISCUSSION

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

This chapter presents the tabulated results of the Butterfat assay in the study

entitled “Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation”

3.1 Mean of Zone of Clearing (ZOC) formed by Isolates A and B.

Fungal Isolate
R1 R2 R3 Mean(Fungal Isolate A)
A

1 20 15 14 16.3333333

2 22 18 30 23.3333333

3 21 25 7 17.6666667

4 11 9 6 8.66666667

5 16 16 10 14

Table 2. Mean of Zone of Clearing formed by Isolate A

Table 1 shows the mean of Zone of Clearing formed by fungal isolate A when

subjected to butterfat assay. It shows that isolate 2 has the highest mean of zone of clearing

with a mean of 23.3333333. Isolate 4 has the lowest mean of zone of clearing with the value

of 8.66666667. Isolate 1 has a mean of 16.3333333; Isolate 3 has a mean 17.6666667 and

isolates 5 with a mean of 14. The result suggests that fungal isolates A have the capability to

degrade oil.

Fungal Isolates
R1 R2 R3 Mean(White)
B

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

6 17 23 15 18.3333333

7 15 16 14 15

8 19 25 21 21.6666667

9 16 18 20 18

10 33 25 18 25.3333333

Table 3. Zone of Clearing (ZOC) formed by Isolate B

Table 2 shows the mean of Zone of Clearing formed by fungal isolate B when

subjected to butterfat assay. It shows that isolate 10 has the highest mean of zone of clearing

with a mean of 25.3333333. Isolate 7 has the lowest mean of zone of clearing with the value

of 15. Isolate 8 has a mean of 21.6666667; Isolate 6 has a mean 18.3333333and Isolate 5 has

a mean of 18. The results suggest that fungal isolates B have the capability to degrade oil.

3.2. Analysis of Variance on the zone of clearing formed by Fungal Isolates A and B

Fungal Isolate Mean P-Value Description

1 16.3333333
2 23.3333333

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

3 17.6666667
4 8.66666667
5 14 0.106883576 Not Significant
6 18.3333333
7 15
8 21.6666667
9 18
10 25.3333333

Table 4 F-test ANOVA of Zone of Clearing formed by Fungal Isolates A and B

Table 4 shows the F-test ANOVA for Zone of Clearing formed by Fungal Isolates A

and B. The table shows that the P value is 0.106883576. Since P value<0.05, the result shows

that there is no significant difference of the Zone of Clearing formed by Fungal Isolates A

and B. Therefore, the fungal isolates A and B have the same capability to degrade oil.

CHAPTER 4

CONCLUSIONS AND RECOMMENDATION

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

This chapter presents the summary of findings, conclusion and recommendations of the

experimentation.

4.1 Summary

Findings

1. On fungal isolate A, isolate # 2 has the highest mean of Zone of Clearing with the

value of 23.33333, followed by isolate #3 with a mean of 17.6666667, isolate #1 has

a mean of 16.3333333, isolate #5 has a mean of 14 and the lowest mean of zone of

clearing in isolate is isolate #4 with a mean of 8.66666667

2. On fungal isolate B, isolate # 10 has the highest mean of Zone of Clearing with the

value of 25.33333333, followed by isolate #8 with a mean of 21.66666667, isolate #6

has a mean of 18.33333333, isolate #9 has a mean of 18 and the lowest mean of zone

of clearing is isolate is isolate #7 with a mean of 15

3. The F-test ANOVA shows that Sig<0.05. Therefore, there is no significant difference

between the zone of clearing of oil spill when treated with Fungal Isolates A and B

4.2 Conclusions

Based on the findings above, the following conclusions are drawn:

29
 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

1. Fungal Isolates A and B from oil contaminated soil can degrade oil.

2. Fungal Isolates A and B have the same capability in degrading oil.

4.3 Recommendations

As synthesized from the findings and conclusions, the following

recommendations were made:

1. Determine the turbidity of the oil spill when treating using fungal isolates.

2. Determine the characteristics of the fungal isolates used in oil degradation.

3. Test the amount of lipase produced by the fungal isolates.

Bibliography:

April, T. M., Foght, J. M., & Currah, R. S. (1999). Hydrocarbon-degrading filamentous fungi
isolated from flare pit soils in northern and western Canada. Canadian Journal of

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

Microbiology, 46(1), 38-49. Retrieved from:

http://www.nrcresearchpress.com/doi/abs/10.1139/w99-117#.XDzRj1UzbDc

Cerniglia, C. E., & Perry, J. J. (1973). Crude oil degradation by microorganisms isolated
from the marine environment. Zeitschrift für allgemeine Mikrobiologie, 13(4), 299-
306.Retrieved from: https://onlinelibrary.wiley.com/doi/abs/10.1002/jobm.19730130403

El Azzabi, T. S., Clarke, J. H., & Hill, S. T. (1981). Lipolytic activity of fungi on rapeseed
oil. Journal of the Science of Food and Agriculture, 32(5), 493-497.Retrieved from:
https://onlinelibrary.wiley.com/doi/abs/10.1002/jsfa.2740320512

Fedorak, P. M., Semple, K. M., & Westlake, D. W. S. (1984). Oil-degrading capabilities of

yeasts and fungi isolated from coastal marine environments. Canadian journal of
microbiology, 30(5), 565-571.Retrieved from:
http://www.nrcresearchpress.com/doi/abs/10.1139/m84-085#.XDy1CVUzbDc

Gopinath, S. C., Anbu, P., & Hilda, A. (2005). Extracellular enzymatic activity profiles in
fungi isolated from oil-rich environments. Mycoscience, 46(2), 119-126.Retrieved from:
https://link.springer.com/article/10.1007/s10267-004-0221-9

Husaini, A., Roslan, H. A., Hii, K. S. Y., & Ang, C. H. (2008). Biodegradation of aliphatic
hydrocarbon by indigenous fungi isolated from used motor oil contaminated sites. World
Journal of Microbiology and Biotechnology, 24(12), 2789-2797.Retrieved from:
https://link.springer.com/article/10.1007/s11274-008-9806-3

Ijah, U. J. J. (1998). Studies on relative capabilities of bacterial and yeast isolates from
tropical soil in degrading crude oil. Waste Management, 18(5), 293-299.Retrieved from:
https://www.sciencedirect.com/science/article/pii/S0956053X98000373

Jaouani, A., Guillén, F., Penninckx, M. J., Martínez, A. T., & Martínez, M. J. (2005). Role of
Pycnoporus coccineus laccase in the degradation of aromatic compounds in olive oil mill
wastewater. Enzyme and Microbial Technology, 36(4), 478-486.Retrieved from:
https://www.sciencedirect.com/science/article/pii/S014102290400331X

Kinderlerer, J. L. (1994). Degradation of the lauric acid oils. International biodeterioration

& biodegradation, 33(4), 345-354.Retrieved
from:https://www.sciencedirect.com/science/article/pii/0964830594900124

Mancera-Lopez, M. E., Rodriguez-Casasola, M. T., Rios-Leal, E., Esparza-Garcia, F.,

Chavez-Gomez, B., Rodriguez-Vazquez, R., & Barrera-Cortes, J. (2007). Fungi and Bacteria
Isolated from Two Highly Polluted Soils for Hydrocarbon Degradation. Acta Chimica
Slovenica, 54(1).Retrieved from: http://acta-arhiv.chem-soc.si/54/54-1-201.pdf

31
 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

Rudd, L. E., Perry, J. J., Houk, V. S., Williams, R. W., & Claxton, L. D. (1996). Changes in
mutagenicity during crude oil degradation by fungi. Biodegradation, 7(4), 335-343.Retrieved
from: https://link.springer.com/article/10.1007/BF00115747

Sorkhoh, N. A., Ghannoum, M. A., Ibrahim, A. S., Stretton, R. J., & Radwan, S. S. (1990).
Crude oil and hydrocarbon-degrading strains of Rhodococcus rhodochrous isolated from soil
and marine environments in Kuwait. Environmental Pollution, 65(1), 1-17.Retrieved from:
https://www.sciencedirect.com/science/article/pii/0269749190901626

Yateem, A., Balba, M. T., Al-Awadhi, N., & El-Nawawy, A. S. (1998). White rot fungi and
their role in remediating oil-contaminated soil. Environment International, 24(1-2), 181-
187.Retrieved from: https://www.sciencedirect.com/science/article/pii/S0160412097001347

APPENDIX A

Letter for Validation

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 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

AMOR R. LALISAN
Science Teacher
Agusan Del Sur National High School

Sir/Maam:
Greetings! The undersigned are presently conducting a research study entitled, “Lipolytic
Fungi Isolates from Oil Contaminated Soil for Oil Spill Degradation”. This is a partial
fulfillment of the requirements of the subject Research Capstone. In this connection the
undersigned humbly request you to review, critique, and validate the content of the procedure
to be used in the study. Please rate each procedure in the criterion below:
CRITERION: The item is suitable to evaluate the problem of the study.
Please evaluate each survey item using the rating scale below:
5- The item is very appropriate to determine if the Lipolytic Fungi from Oil
Contaminated Soil can degrade oil.
4- The item is appropriate to determine if the Lipolytic Fungi from Oil Contaminated
Soil can degrade oil.
3- The item is appropriate but needs improvement;
(Please indicate the needed improvement under the remarks.)
2- The item as it is written, is not appropriate: it should be revised;
(Please write the suggested revision under the remarks.)
1- The item is totally inappropriate; discard.
For your generosity of times and graciousness in sharing your experience, the researchers
would like to thank you very much!
Respectfully yours,

MARC LOWELL VILLOTE LORAINE PIMENTEL

DAREEN JAN ROMERO QUENNIE CHILL CARBONELL
DANIELLEE EUNICE DOCUAN
PRINCE BO LASTIMOSA

Noted by:
JEFFREY Q. APAT
Research Adviser

JENNYVI H, PAPELLERO
Research Adviser

JUN REY M. MEKING

Research Teacher
Agusan Del Sur National High School

33
 Fungal Isolates from Oil Contaminated Soil for Oil Spill Degradation

Sir/Maam:
Greetings!
The undersigned are presently conducting a research study entitled, “Lipolytic Fungi
Isolates from Oil Contaminated Soil for Oil Spill Degradation”. This is a partial
fulfillment of the requirements of the subject Research Capstone. In this connection the
undersigned humbly request you to review, critique, and validate the content of the procedure
to be used in the study. Please rate each procedure in the criterion below:
CRITERION: The item is suitable to evaluate the problem of the study.
Please evaluate each survey item using the rating scale below:
5- The item is very appropriate to determine if the Fungi from Oil Contaminated Soil
can degrade oil.
5- The item is appropriate to determine if the Fungi from Oil Contaminated Soil can
degrade oil.
4- The item is appropriate but needs improvement;
(Please indicate the needed improvement under the remarks.)
3- The item as it is written is not appropriate: it should be revised;
(Please write the suggested revision under the remarks.)
2- The item is totally inappropriate; discard.
For your generosity of times and graciousness in sharing your experience, the researchers
would like to thank you very much!
Respectfully yours,

The Researchers

Noted by:

JEFFREY Q. APAT
Research Adviser

JENNYVI H, PAPELLERO
Research Adviser

34