- Faculty of Biosciences and Medical

i
“I hereby declare that I have read this thesis and in my opinion this thesis is
sufficient in terms of scope and quality for the award of Bachelor of Science
(Biology)”
Signature
: …………………………………………………
Name
: DR. TOPIK HIDAYAT
Date
: 6TH JULY 2012
MOLECULAR IDENTIFICATION OF FUNGUS THAT CAPABLE TO
DEGRADE PERSISTENT ORGANIC POLLUTANT
JULIANA NATASHA BINTI JAMALUDIN
A report submitted in partial fulfilment of the
requirement for the award of the Degree of
Bachelor of Biology (Science)
Faculty of Biosciences and Bioengeneering
Universiti Teknologi Malaysia
MAY 2012
ii
I declare that this thesis entitled Molecular Identification of Fungus That Capable to
Degrade Persistent Organic Pollutants (POPs) is the result of my own research
except as cited in the references. The thesis has not been accepted for any degree and
is not concurrently submitted and candidature of any other degree.
Signature
: …………………………………………..
Name
: Juliana Natasha Binti Jamaludin
Date
: 6th July 2012
iii
To my beloved father and mother and my lovely siblings
iv
ACKNOWLEDGEMENTS
In the name of Allah, the Most Gracious and the Most Merciful.
Alhamdulillah, all praises to Allah for the strengths and His blessing in
completing this thesis. This thesis would not have been possible without the guidance
and help from several individuals who contributed directly or indirectly and extended
their valuable assistance in the preparation and completion of this study.
First and foremost, my utmost gratitude to my supervisor, Dr. Topik Hidayat
whose sincerity and encouragement I will never forget. Dr. Topik has been my
inspiration as I hurdle all the obstacles in the completion this research work. I
attribute to his encouragement and effort and without him this thesis, too, would not
have been completed or written. One simply could not wish for a better or friendlier
supervisor.
I would like to express my appreciation to senior, Mohd Atiq, Chandrika,
Farah Izana, Mohd Safwan and also Tengku Idzzan for their support and help
towards my final year undergraduate project. My acknowledgement also goes to all
the technicians and office staffs of Faculty Biosciences and Bioengineering for their
co-operations. Sincere thanks to all my friends especially Farhanah, Hidayah,
Sharifah, Ryan, Ragheed and others for their kindness and moral support during my
study. Thanks for the friendship and memories.
Last but not least, my deepest gratitude goes to my beloved father; Mr.
Jamaludin Bin Jantan and also to my siblings for their endless love, prayers and
encouragement. To those who indirectly contributed in this research, your kindness
means a lot to me. Thank you very much.
v
ABSTRACT
Persistent organic pollutant is one of the chemical that has been widely used
around the world. However, this chemical is proven to be hazardous to human health
and the environment and becoming the greatest problems of modern world-society.
These compounds naturally degrade very slowly, so they remain in the environment
for a long time. In this research, a fungi isolated from Hutan Rekreasi UTM were
identified by molecular approach using 18s rDNA, which include 18s rRNA gene
sequence and also the phylogenetic tree construction. The 18S rRNA gene were
being isolated and amplified by Polymerase Chain Reaction (PCR) using universal
primer, NS1 as forward primer and NS8 as the reverse primer from sample named
S7. Next, the PCR product were sequenced by using tools for nucleotide which is
Basic Local Alignment Tool for nucleotide (BLASTn) and being compared with the
sequences retrieved from GeneBank database of National Center of Biotechnology
Information (NCBI). The sequence then was analyzed and phylogenetic genetic tree
were constructed by using software called Molecular Evolutionary Genetic Analysis
(MEGA) version 5.0 to describe and show us the similarity of the sample’s sequence
with the other fungi in the database sequence. The BLASTn analysis shows that S7
was closely related to Meyerozyma guilliermondii (JN546137), given the highest
similarity to the database. In conclusion, Meyerozyma guilliermondii can be used in
degrading
persistent
organic
pollutant
in
the
environment.
vi
ABSTRAK
Bahan-bahan pencemar organik persisten merupakan salah satu bahan kimia
yang digunakan secara meluas di serata dunia. Walau bagaimanapun, bahan kimia ini
telah menjadi masalah terbesar bagi masyarakat kini kerana ia telah terbukti
membahayakan kesihatan manusia sejagat dan juga alam sekitar. Secara semula
jadinya, bahan-bahan kimia ini terurai dengan perlahan menyebabkan ia bertahan
lama di dalam alam sekitar. Dalam kajian ini, kulat yang diambil daripada Hutan
Rekreasi UTM telah dikenal pasti menggunakan pendekatan 18S rRNA, melalui
penjujukan gen dan pembinaan pokok filogenetik. Gen 18S rRNA dipencilkan
daripada sampel kulat yang dilabel sebagai S7 melalui Polymerase Chain Reaction
(PCR) menggunakan primer NS1 dan NS8. Produk PCR dirangkai dan dibandingkan
dengan jujukan yang terdapat di dalam pangkalan data Bank Gen dari National
Center of Biotechnology Information (NCBI) dengan menggunakan perician carian
untuk nukleotida iaitu Basic Local Alignment Search Tool for nucleotide (BLASTn).
Urutan yang telah dirangkai ini juga dianalisis dan pokok filogenetik dibina
menggunakan perisian Analisis Genetik Evolusi (MEGA) versi 5.0 untuk
menunjukkan kesamaan yang terdekat dengan jujukan DNA dari pangkalan data.
Keputusan analisis BLASTn menunjukkan sampel S7 berkait rapat dengan
Meyerozyma
guilliermondii
(JN546137).
Kesimpulannya,
Meyerozyma
guilliermondii boleh digunakan untuk merungkai bahan-bahan pencemar organik
persisten dalam alam sekitar.
vii
TABLE OF CONTENTS
CHAPTER
1
2
3
TITLE
PAGE
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENTS
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
ix
LIST OF FIGURES
x
INTRODUCTION
1
1.1
Problem Background
1
1.2
Problem Statement
2
1.3
Objectives of Study
2
1.4
Scope of Study
3
1.5
Research Significance
3
LITERATURE REVIEW
5
2.1
Persistent Organic Pollutants: A Global Issue
5
2.2
Fungi in Bioremediation
10
2.3
Phylogenetic Tree
12
MATERIALS AND METHODS
14
3.1
Experimental Design
14
3.2
Preparation of Materials
15
3.2.1 Preparation of Potato Dextrose Broth (PDB)
15
3.2.2 Preparation of 10X TAE Buffer
15
viii
3.2.3 Preparation of 1%(w/v) Agarose Gel
15
3.3
Fungi Sampling
17
3.4
Extraction of Fungal DNA
17
3.5
Polymerase Chain Reaction (PCR)
18
3.6
PCR Product Purification
20
3.7
DNA Sequencing
21
3.8
Basic Local Alignment Search Tool for Nucleotide
21
(BLASTn)
3.9
4
Phylogenetic Tree Construction
21
RESULTS AND DISCUSSION
22
4.1
Culturing Fungi in Potato Dextrose Broth (PDB)
22
4.2
Gel Electrophoresis for Genomic Extraction
23
4.3
PCR Result
24
4.4
DNA Purification
25
4.5
DNA Sequencing
25
4.6
Homology Similarity Search By Using Basic Local
26
Alignment Search Tool for Nucleotide (BLASTn)
5
4.7
Multiple Sequence Alignment
28
4.8
Phylogenetic Tree Construction
28
CONCLUSIONS AND FUTURE WORKS
30
5.1
Conclusion
30
5.2
Future Works
31
REFERENCES
32
APPENDIX
37
Appendix A
37
Appendix B
40
ix
LIST OF TABLES
TABLE NO.
TITLE
PAGE
3.1
Amount of components used in PCR reaction.
19
3.2
Thermal cycle profile for PCR reaction of 18S rRNA.
20
3.3
Primers used for 18S rRNA amplification and
20
sequencing.
4.1
Purification result of sample S7 by using nanodrop.
25
x
LIST OF FIGURES
FIGURE NO
2.1
TITLE
Structural formula of polychlorinated biphenyls (PCB),
PAGE
6
C12H(10-n)Cln.
2.2
Structural formula of polychlorinated dibenzo-p-dioxins
6
(PCDD), C12N(8-n)ClnO2 , n=1-8
2.3
Structural formula of polychlorinated dibenzofurans
7
(PCDF), C12H(8-n)ClnO, n=1-8
2.4
Structural formula of aldrin, C12H8Cl6
8
2.5
Structural formula of dieldrin, C12H8Cl6O
8
2.6
Structural formula of endrin, C12H8Cl6O
9
2.7
Structural formula of chlordane, C10H6Cl8
9
2.8
Structural formula of heptachlor, C10H5Cl7
10
2.9
Structural formula of Dichlorodiphenyltrichloroethane
10
(DDT), C14H9Cl5
2.10
Universal phylogenetic tree shows the three major
12
domains.
2.11
Detailed tree for eukaryotic shows the trunk branching to
13
kingdoms.
2.12
Detailed tree for the archaea domain.
13
3.1
Flow chart for identification of fungi using molecular
14
approach.
3.2
1kb Promega Ladder
17
4.1
Fungi sample 14 days after culture in Potato Dextrose
22
Broth (PDB).
xi
4.2
Analysis of genomic extraction of sample S7 using 1%
23
(w/v) agarose gel electrophoresis.
4.3
Analysis of polymerase chain reaction (PCR) of the
24
amplified fragment from fungi sample S7 in 1% (w/v)
agarose gel electrophoresis
4.4
NS1 sequence for sample S7
26
4.5
Blast result for sample S7.
27
4.6
Phylogenetic tree of sample S7
29
1
CHAPTER 1
INTRODUCTION
1.1.
Problem Background
Throughout the past century, industrial, military, and farming activities have
released many organopollutants into the environment. Since World War II, there
have been thousands of chemicals introduced for commercial use. Most of these
chemicals have been really helpful in controlling the pest and disease, and have
increase crop production and industry. Persistent organic pollutant is one of the
chemicals that has been widely used around the world. However, this chemical has
been proven to be hazardous to human health and the environment thus becoming the
greatest problems of modern world-society. These compounds naturally degrade very
slowly, so they remain in the environment for a long time.
The most important and effective way to remove the compound contained in
the pesticides from the environment is by using biological decomposition.
Microorganisms have the capability to interact, both chemically and physically, with
substances leading to changes in the structure or absolute degradation of the target
molecule (Raymond et al., 2001; Wiren-Lehr et al., 2002). The main transformers
and pesticides degraders include bacteria, fungi and actinomycetes (De Schrijver and
De Mot, 1999).
The primary focus of this review is to identify fungi that can undergo
xenobiotic degradation to help in degrading the POPs as the traditional waste
disposal methods such as incineration and landfilling need a longer time to degrade
2
it. The fungi used in this experiment will also be characterize based on its molecular
to construct a phylogenetic tree to help us know which type of fungal group is
suitable in the degrading the POPs
1.2
Problem Statement
Humans can be exposed to POPs through food, accidents and the
environment (including indoors). Exposure to POPs, either acute or chronic, can be
associated with a wide range of adverse health effects, including illness and death
(Siripong, 2009). Laboratory investigations and environmental impact studies in the
wild have implicated POPs in endocrine disruption, reproductive and immune
dysfunction, neurobehavioural and disorders and cancer (Ritter, 1995). More recently
some POPs have also been implicated in reduced immunity in infants and children,
and the concomitant increase in infection, also with developmental abnormalities,
neurobehavioural impairment and cancer and tumour induction or promotion. Some
POPs are also being considered as a potentially important risk factor in the etiology
of human breast cancer
It is a cheaper method to decontaminate toxic waste sites in the United States
rather than using traditional waste disposal methods such as incineration and landfill
sites (Reddy and Mathew, 2001) Due to this problem and the need of a reasonable
solution, a quick, cost-effective, ecologically responsible method of cleanup is really
needed. One of the mechanisms of decontamination that may be suitable for all these
requirements is bioremediation (Sarah, 2004)
1.3
Objectives of Study
The objectives of this study include:
3
a) To identify fungus that is capable of degrading persistent organic pollutant
(POP).
b) To construct a phylogenetic tree of the isolated fungi.
1.4
Scope of the Study
The scope of the study includes two main parts which excludes an in vivo
samples provided by Dr Tony from EPASA, UTM which was collected in Hutan
Rekreasi UTM. The first part is in vitro a study which covers the method of culturing
the fungi; genomic extraction by using Wizard Genomic Purification Kit by
Promega, polymerase chain reaction by using i-taq DNA Polymerase kit from
Intronbio and the use of gel electrophoresis.
The second part of this study is in silico which is bioinformatic data analysis.
The sequence that is being extracted will be sent to the gene bank to be analysed.
After receiving the result from the gene bank, further analysis will be done by using
basic local alignment search tool (BLAST) and MEGA to know the fungi species and
be able to construct the phylogenetic tree to compare the fungi genetically.
1.5
Research Significance
Knowing the fungi that can degrade the persistent organic pollutant (POPs)
will help us in controlling the pollution and reduces the effects on human health in
many ways. Microbial degradation is an environmentally friendly and a cost
competitive alternative to physico-chemical decomposition processes for the
treatment of industrial effluents.
4
It is recommended that degradation products should be included in hazard
assessments to gain a more accurate insight into the environmental risk of chemicals.
The result of this project could also be combined with information on the toxicity of
the degradation products. This should be helpful to provide further insight into the
significance of degradation products for environmental hazard assessments.
5
CHAPTER 2
LITERATURE REVIEW
2.1
Persistent Organic Pollutants: A Global Issue.
Persistent Organic Pollutants (POPs) can be described as chemical
compounds that resist photolytic, chemical, and biological degradation. POPs are
usually halogenated and can be characterised by high lipid solubility and low water
solubility which will lead to the bioaccumulation in fatty tissues (Cumanova et al.,
2007). POPs can be catagorized as a toxic substance as well as being prone to longrange transport (Nostrom et al., 1988). According to Hailong (2010), POPs can be
classified into three categories: (1) industrial chemical product such as
polychlorinated byphenils (PCBs); (2) Unintentionally formed by products such as
hexachlorobenzene
(HCB),
polychlorinated
dibenzo-p-dioxins
(PCDD),
polychlorinated dibenzo-p-furans (PCDF) and polychlorinated byphelis (PCBs); (3)
and pesticides such as aldrin, dieldrin, endrin, mirex, toxaphene, hexaclorobenzene,
heptachlor, chlordane and dichlorodiphenyl trichloroethane (DDT).
Industrial chemicals was first discovered as environmental pollutants in 1966,
polychlorinated biphenyls or PCB compound as in Figure 2.1 have been found
throughout the world in various places such as water, sediments, bird tissue and also
fish tissue (El-Shawawi et al.,2009). It is mainly used as additives and also in
electrical equipment for example in lubricants, adhesives and hydraulic fluids (Vit,
1992). PCBs are a class of chlorinated hydrocarbons that consist of two benzene
rings joined by a carbon-carbon bond, with chlorine atoms substituted on any of all
the remaining 10 carbon atoms.
6
Figure 2.1 Structural formula of polychlorinated biphenyls (PCB), C12H(10-n)Cln.
Unintentionally POPs are the by-products formed from combustion or
chemical process that take places when chlorine compounds are present.
Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans
(PCDF) are unintentionally formed of persistent organic pollutant whereas dioxins
and furans are chemical substances that are released by a thermal processes which
involves organic matter together with chlorine resulting from incomplete chemical
reactions and combustion. PCDDs and PCDFs consist of tricycle aromatic
compounds, two benzene ring connected by two oxygen atoms (for PCDDs) or one
oxygen atom (for PCDFs) whiles the hydrogen atoms can be replaced by a maximum
8 chlorine atoms. Figure 2.2 and 2.3 shows the structural formula of polychlorinated
dibenzo-p-dioxins (PCDD) and Structural formula of polychlorinated dibenzofurans
(PCDF) respectively.
Figure 2.2
Structural formula of polychlorinated dibenzo-p-dioxins (PCDD),
C12N(8-n)ClnO2 , n=1-8
7
Figure 2.3
Structural formula of
C12H(8-n)ClnO, n=1-8
polychlorinated
dibenzofurans
(PCDF),
Physically, dioxins exists as colourless compound while furans exist as nonodorous white-needle shaped crystals. Both PCDD (Figure 2. 2) and PCDF (Figure
2.3) were shown to cause developmental toxicity, cancers, mutagenecity and
endocrine disruption to animals and most of all to humans. (Eljarrat et al., 2004)
Intentionally POP compounds were produced which lead to a reaction with
chlorine. These types of compound have high lipopolicity, usually high neurotoxicity
and organic molecules that link to chlorine atoms called organochlorine compounds
(OCs). Chlorinated insecticides such as dichlorodiphenylchloroethane (DDT) are one
example of OCs. Other than that, chemical that can be classified under POPs
pesticides are aldrin, dieldrin, endrin, mirex, toxaphene, hexachlorobenzene,
heptachlor and chlordane.
Aldrin as in figure 2.4 can be characterized as white and odourless crystals
when they are pure. It can be either non-corrosive or slightly corrosive to certain
metals. Aldrin is readily converted to dieldrin in the environment and is widely used
to protect crops. Aldrin is being used to control soil insects such as termites, corn
rootworm and grasshoppers. Although aldrin is effectively used to protect wooden
structures from termites, the presence of aldrin is toxic to humans.
8
Figure 2.4
Structural formula of aldrin, C12H8Cl6
Dieldrin as in figure 2.5 is closely related to aldrin as it is the stereoisomer of
the aldrin. It might be present as white crystals or pale tan flakes and odourless. The
used of dieldrin is just as important as aldrin. Dieldrin residues can be detected in
water, soil, air, fish, birds and mammals including human breast milk. Chemically,
dieldrin has a low phototoxicity.
Figure 2.5
Structural formula of dieldrin, C12H8Cl6O
Figure 2.6 is referred to endrin which is a white odourless crystalline solid
when pure and exists as a stereoisomer of dieldrin. Endrin is an organochlorine foliar
insecticide that has been used to control most of the agricultural pests mostly on
cotton, rice, sugarcane and grain. It also acts as a rodenticide to control cutworms,
mice and borers. Endrin is highly persistent in soil and rapidly metabolised by
animals and does not accumulate in fat like other compound with similar structure. It
is highly toxic to fish, aquatic invertebrates and phytoplankton.
9
Figure 2.6
Structural formula of endrin, C12H8Cl6O
Chlordane which can be referred to in Figure 2.7 can be seen as a colourless
to amber-coloured viscous liquid with a pungent smell similar to chlorine. It has been
widely use as an insecticide to control the population of cockroaches, ants, termites
and other pests. It’s been detected in arctic air, water and organism.
Figure 2.7
Structural formula of chlordane, C10H6Cl8
Based on figure 2.8, heptachlor is a white to light tan compound, waxy solid
or crystals with a camphor-like odour. It is a nonphytotoxic at insecticidal
concentrations and persistent dermal insecticides with some fumigant action. It is
used in soil and seed treatment to protect corn and small grains from pest. Heptachlor
is highly insoluble in water and soluble in organic solvent and also very toxic to
mammals.
10
Figure 2.8
Structural formula of heptachlor, C10H5Cl7
Dichlorodiphenyltrichloroethane (DDT) as in Figure 2.9 appears as an
odourless colourless crystal or white powder. It was widely used during the World
War II to protect troops and civilians from the spread of vector borne diseases such
as malaria and thyphus. DDT is highly insoluble in water and is soluble in most
organic solvents. DDT is highly toxic to fish, acutely toxic to birds and in the human
organism; DDT exhibits oestrogen-resembling and probable carcinogenic properties.
Figure 2.9
2.2
Structural
C14H9Cl5
formula
of
Dichlorodiphenyltrichloroethane
(DDT),
Fungi in Bioremediation
Fungi are part of the eukaryotic family, they include microorganisms such as
yeast, moulds and mushrooms. These organisms are categorized as a kingdom,
Fungi, is totally different from plants, animals, and bacteria. One major difference
between fungi and plants is that fungal cells have cell walls that made up of chitin,
unlike that of the cell wall of plants, which contain cellulose (Bowman, 2006). Fungi
comprise of a variety of microorganisms and are numerically among the most
11
abundant eukaryotes on earth’s biosphere. Most fungi can be found in soil, on dead
matter, and as symbionts of plants, animals, or other fungi. Fungi perform a crucial
role in the breakdown of organic matter and have important roles in nutrient
cycling and exchange.
Bioremediation is defined as the purpose of biological processes as a
treatment to pollution. Most research within the field of bioremediation has focused
on bacteria, with fungal bioremediation (mycoremediation) attracting concern within
the past two decades. Processes of natural bioremediation of lignocelluloses occupy
a variety of organisms, but predominantly involving fungi as white rot fungi as it can
withstand toxic levels of most organopollutants (Weinstock, 1997 and Aust et al.,
2004). The ability of a fungus to facilitate remediation of contaminated environment
is becoming an intense interest as it offers a low-cost and ecologically acceptable
approach to dissipating pollutants (Anderson, 1993).
Many ectomycorrhizas fungi produce POP-degrading enzyme such as
laccase, tyrosinasses, catechol oxidases, manganese peroxidises (MNPs), and lignin
peroxidises (LIPs) (Meharg et. al., 2000). LIPs, MNPs and laccases are three classes
of enzyme that are important in lignin degradation and are stimulated by nutrient
limitation (Mansur et al., 2003; Aust et al., 2004). These three enzymes can be
suppressed completely in the high nitrogen levels media (Reddy et al, 2001).
LIPs (EC 1.11.1.7) are relatively non-specific, which can degrade random
structures of lignin (Reddy, 1995). This characteristic enables the enzyme to oxidize
a variety of xenobiotics compounds that share similar characteristics to lignin
substructures. MNPs (EC 1.11.1.7) are able to oxidize variety phenolic substrate
(Kirk, 1987) whereas laccase (EC 1.10.3.2) utilise molecular oxygen as an oxidant
and also oxidize phenolic substrate to phenoxy radicals (Hatakka, 1994)
12
2.3
Phylogenetic Tree
A phylogenetic tree which is also called as evolutionary tree is a diagram
with branches that shows the evolutionary relationships among various biological
species or other entities based on their similarities and differences in their genetic
characteristics. Phylogeny is based on the concept of genetic evaluation which
depends on additional changes in the chromosomal DNA sequences leading
progressively to new species (Rittmann and McCarty, 2001).
The phylogeny tree is a perceptive that all new species have one evolutionary
progenitor that gives root of phylogenetic tree, and the root branches into species
(Rittmann and McCarty, 2001).
Universal Ancestor
(the “root”)
Figure 2.10
Universal phylogenetic tree shows the three major domains adapted
from Rittmann and McCarty, 2001.
The phylogenetic tree explains that all living organisms on earth can be
divided into three major domains, which are bacteria, archaea and eukarya. The two
domains which are archaea and bacteria are grouped as prokaryotes and these
domains are at the trunk of the phylogenetic tree (Rittmann and McCarty, 2001).
13
Figure 2.11
Detailed tree for eucaryoticshows the trunk branching to kingdoms
(Rittmann and McCarty, 2001).
Every kingdom is separated into smaller branches that gives details between
related species. The liner space between related species and others along the tree
quantifies their genetic distance. Figure 2.5 shows the close relationship between
plants and fungi (Rittmann and McCarty, 2001).
Figure 2.12
Detailed tree for the archaea domain (Rittmann and McCarty, 2001).
The universal presence of small subunit rRNA become the standard data for
molecular polygenetic of fungi diagnosis (Khachatourians and Arora, 2003).
14
CHAPTER 3
MATERIALS AND METHODS
3.1
Experimental Design
The experiment involves both in vitro and in silico parts, in vitro meaning
that this part of the experiment is being done inside the laboratory, whilst in silico is
using bioinformatics analysis needed to prove the experiments theory.
Fungi Sampling
Agarose Gel
Electrophoresis
Purification of PCR
Product
Preparation of
Potato Dextrose
Broth (PDB)
Polymerase Chain
Reaction (PCR)
DNA Sequencing
Agarose Gel
Electrophoresis
Homology
Similarity Search by
BLASTn and
Phylogenetic Tree
analysis
Genomic Extraction
Figure 3.1
Flow chart for identification of fungi using molecular approach.
15
3.2
Preparation of Materials
3.2.1
Preparation of Potato Dextrose Broth (PDB)
Potato dextrose broth is a common microbiological growth media made from
potato infusion and dextrose. Potato dextrose broth is the most widely used medium
for growing fungi and bacteria which attack living plants or decaying dead plant
matter.
All fungal strains were freshly cultured aerobically in PDB (1.2g in 50ml
distilled water) at 30°C for two weeks until they reached full growth before
extraction. Next, the PDB which contain the fungal sample were incubated in the
orbital shaker to maximize their grow
3.2.2
Preparation of 10X TAE Buffer
TAE buffer is a buffer solution that consists of Tris base, acetic acid and
Ethylenediaminetetraacetic acid (EDTA). In this experiment, TAE buffer is used in
agarose gel electrophoresis typically for the separation of nucleic acid such as DNA
and RNA. It is also used as running buffer in garose gel electrophoresis. To prepare
1X TAE buffer from 50X TAE buffer, 20 mL 50x TAE buffer is diluted in 1 liter
distilled water and mix it thoroughly.
3.2.3
Preparation of 1% (w/v) Agarose Gel.
DNA Agarose gels can be used to separate and visualize DNA of various
sizes ranging from 50 base pair to several mega bases using specialized apparatus.
The distance between DNA bands of a given length is determined by the percentage
16
of agarose used for the gel. Firstly 0.5g of agarose gel is weight before mixing it with
50ml Tris-Acetate EDTA (1X TAE buffer) in Erlenmayer flask to form 1% (w/v)
agarose gel. The buffer should not occupy more than 50% of the volume of the flask.
Next, the agarose solution is heated on the hot plate to allow all of the grains of
agarose to dissolve. After all the grains are dissolved, the solution is allowed to cool
to cool down at room temperature for 5 minutes before adding 1µl of Ethidium
bromide (EtBr) into the solution.
Next, the comb is positioned above the plate, thus allowing a complete well
to be formed when agarose solidifies. The agarose solution will then being poured
into the tray and the formation of air bubble should be avoided under or between the
teeth of the comb. After about 30 minutes the gel has solidified, the comb is then
removed and the gel is placed into the electrophoresis tank. 1X TAE buffer is added
into the tank to cover the gel until the top of the well is submerged. 5 µl samples are
mixed with 1µl loading buffer. The mixture is loaded slowly into the well using a
micropipette and run with 90V voltage for 30 minutes. The lid of the tank is closed
and making sure that the samples are placed at the correct end of the apparatus with
respect to the anode and cathode. The unit is working when we see bubbles formed
in the buffer due to the electrical field that has been created; We are then able to see
the dye moving down the gel. After the electrophoresis is complete, the molecules in
the gel can be visible under UV light or can be viewed using UV transiluminator.
Figure 3.2 shows 1kb ladder used in this study.
17
Figure 3.2
3.3
1kb Promega Ladder
Fungi Sampling
The fungi were sampled from Hutan Rekreasi UTM. It was then tested by Dr.
Tony from IPASA, UTM if the fungi were able to degrade persistent organic
pollutants (POPs). After the fungi was proved to be able to degrade POPs, it was then
further analyzed by me to identify the fungi species.
3.4
Extraction of Fungal DNA
DNA extraction is the first step in many protocols when the ultimate
goal is to analyze a portion of the DNA. Careful extraction technique will result in
pure, clean DNA that can be readily analysed. In this experiment we used a simple
extraction method (Al-Samarrai and Schmid, 2000) to extract DNA from unknown
18
fungi. The genomic DNA of sample S7 was extracted using Wizard Genomic DNA
Purification Kit (Promega) according to the manufacturer’s instructions.
Firstly, a small lump of mycelia was added to liquid nitrogen and ground to a
fine powder using a mortar and pestle. Then the powder is put into a 1.5 ml
microcentrifuge tube which contains 600μl of nuclei lysis solution. Next, 300μl of
cell lysis solution is added before the solution mixture is incubated at 65oC for 30
minutes. 5μl of RNAse solution is added to the cell lysate and the tube containing the
mixture is inverted 2-5 times. The sample is then allowed to cool down to room
temperature for 5minutes before the addition of 200μl of protein precipitation
solution; then vortex it vigorously. After that, the microcentrifuge tube containing the
mixture is place in the centrifuge for 3 minutes at 13000rpm. The supernatant which
contains DNA is carefully removed into a new microcentrifuge tube containing
600μl of 100% isopropanol kept at room temperature. The solution is mixed gently
by inversion until thread-like strands of DNA form a visible mass before centrifuge
again for 1 minute at high speed. Next, the supernatant was discarded and 600μl of
70% alcohol was added into the tube to wash the DNA. The tube was centrifuge
again at 13000rpm for another minute. The supernatant was discarded and the tube
was inverted on absorbent paper to air dry the pellet for 15 minutes. 100μl of DNA
rehydration solution was added to the tube. Finally, the tube was incubated at 65oC
for 30 minutes. The DNA was stored at -20oC for further use.
3.5
Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) is a technique in molecular biology to
amplify a single or a few copies of DNA generating a thousand or millions of copies
of particular DNA sequence. The method depends on thermal cycling, consisting of
cycles of repeated heating and cooling of the reaction of DNA melting and enzymatic
replication of the DNA.
19
In this experiment, the DNA was amplified by PCR, using Applied
Biosystem (ABI) thermal cycler. The primer pair NS1 and NS8 was used to amplify
the conserve region. Table 3.1 shows the components and the volume required for
each component used in PCR of 18S rRNA genes from sample S7.
The DNA amplification was carried out by using the Perkin Elmer Gene
AmpR PCR thermocycler for 35 cycles. The steps were indicated in Table 3.2.
The 18S rRNA genes from genomic sample S7 were PCR amplified by using
universal primers, NS1 as the forward primer and NS8 as the reverse primer. The
details of the primers are shown in Table 3.3.
Table 3.1: Amount of components used in PCR reaction
Components
Volume (µl)
10x PCR buffer
4.0
dNTP
4.0
Forward Primer
2.0
Reverse Primer
2.0
Genomic Template
2.0
i-Taq Polymerase
1.0
Distilled water
25.0
Total
40
20
Table 3.2
Thermal cycle profile for PCR reaction of 18S rRNA.
Stage
Temperature ( oC)
Duration
Initial denaturation
94
3 min
Denaturation
94
30 sec
Annealing
55
30 sec
Extension
72
2 min
Final extension
72
10 min
Hold
4
∞
35 cycles
Table 3.3: Primers used for 18S rRNA amplification and sequencing
(White et al., 1991).
Primers
NS1
(forward)
NS8
(reverse)
3.6
Sequences
5’-GTAGTCATAAGCCTTGTCTC-3’
5’-TCCGCAGGTTCACCTACGGA-3’
Molecular Weight
5785
6078
PCR Product Purification
PCR clean up or purification was carried out by using QIAquick PCR
Purification Kit from Qiagen according to the manufacturer’s instruction and the
concentration of the PCR product was checked using the Nanodrop instrument. 100
µl of PB buffer was added into 20µl of PCR sample and mixed. The QIAquick spin
column was placed in a provided 2ml collection tube. The sample was applied to the
QIAquick column and centrifuge for 60 second to bind the DNA. The flow through
is being discarded and QIAquick column was being placed back into the same tube.
0.75ml buffer PE was added to the QIAquick column before centrifuge it for another
60 second. Flow through was being discarded and the QIAquick column was placed
back in the same tube and the column was being centrifuge for another minute at
maximum speed. Next, the QIAquick column was placed into a clean 1.5 ml
microcentrifuge tube. 50 µl of buffer EB was added to the center of the QIAquick
membrane and centrifuge the column for one minute to elute the DNA.
21
3.7
DNA Sequencing
The PCR product of 18s rRNA gene from sample S7 as well as the universal
primer which is NS1 and NS8 were sent to 1st BASE Laboratories Sdn. Bhd.,
Malaysia for DNA sequencing. The samples were applied to Biosystem analyzer by
capillary electrophoresis sequencing.
3.8
Basic Local Allignment Search Tool for Nucleotide (BLASTn)
After the PCR product was sequenced, Basic Local Alignment Search Tool, or
BLAST was used to determine the species of the fungi that we had extract. BLAST
is an algorithm for comparing primary biological sequence information, such as the
amino-acid sequences of different proteins or the nucleotides of DNA sequences. A
BLAST search enables a researcher to compare a query sequence with a library
or database of sequences, and identify library sequences that resemble the query
sequence above a certain threshold.
First Base will give us the sequence of the DNA in FASTA format.
Electropherogram is used to determine whether the sequence is good or not before
being modified and key in into the software. The software also could help us in
establishing phylogeny, construct DNA mapping and can locate common genes in
two related species.
3.9
Phylogenetic Tree Construction
The full length of DNA sequence of sample S7 was compared with the
nucleated sequences from Genbank Database and a Phylogenetic Tree was
constructed using Molecular Evolution Genetic Analysis software (MEGA) version
5.0.
22
CHAPTER 4
RESULTS AND DISCUSSIONS
4.1.
Culturing Fungi in Potato Dextrose Broth
After 2 weeks of incubation, it can be seen on Figure 4.1 that S7 fungi grew
well in Potato Dextrose Broth (PDB). The reason why potato dextrose broth is was
chosen as the medium to culture the unknown fungi (S7) is because PDB is a general
medium for yeast and molds that made up of potato infusion and dextrose. The
growth of bacteria is inhibited by the low pH number. It is easy to handle the fungi
since it grew properly in the PDB.
Figure 4.1
Fungi sample 14 days after culture in Potato Dextrose Broth (PDB).
23
4.2.
Gel Electrophoresis for Genomic Extraction
The genomic DNA from fungi sample S7 was isolated using Wizard
Genomic DNA Purification kit (Promega). The isolated fungal genomic DNA was
stored at -20oC prior to study. The purified DNA was examined by using agarose gel
electrophoresis and observed under UV illumination.
Figure 4.2 shows the isolated genomic DNA of sample S7 with five
replicates. The band of genomic DNA of sample S7 which is more than 10kb can be
seen at lanes 2 to 6 where as lane 1 was loaded with the DNA ladder (1kb). Based on
previous research made by Saad et al., 2004, he stated that the size of genomic DNA
isolation of a fungi should be more 10000 base pairs which means the sample band
should be above the 10000 base pairs ladder band.
Ladder (1kb)
S7
S7
S7
S7
S7
10000 bp
Figure 4.2
Analysis of genomic extraction of sample S7 using 1% (w/v) agarose
gel electrophoresis.
24
4.3
PCR Result
18S rRNA from the fungi sample S7 was amplified by PCR using universal
primer; NS1 and NS8. The primers contain of 20 nucleotides and were reported to
amplify almost the full 18S rRNA from fungi (White et al., 1991)
Based on previous research done by Ragheed, 2011, he stated that the
expected length of sequence being amplified by using primer NS1 and NS8 should
be about 1800 base pairs. The amplified fragment was analyzed using agarose gel
electrophoresis and being observed under UV illumination. This was shown on
Figure 4.3 and the 18S rRNA genes were successfully amplified by NS1 and NS8
primers.
Ladder (1kb)
2000bp
1500bp
Figure 4.3
S7
S7
S7
S7
S7
1800bp
Analysis of polymerase chain reaction (PCR) of the amplified
fragment from fungi sample S7 in 1% (w/v) agarose gel
electrophoresis.
25
4.4
DNA Purification
For the PCR purification using QIAquick PCR Purification Kit, Table 4.1
shows that the sample preparation requirement that is needed by to be fulfil before
sending the sample to 1st BASE Laboratories Sdn. Bhd., Malaysia for DNA
sequencing.
As the PCR product obtained from this study was 1800 base pair, so the
sample concentration should be exceeded 40ng/µL. Table 4.1 shows that the result of
sample S7 by using nanodrop method.
Table 4.1 Purification result of sample S7 by using nanodop.
Sample
260/280
260/230
Ng/µl
S7
1.82
1.91
181.7
Based on Table 4.1, it shows that the concentration of the sample S7 had
fulfilled the sequencing concentration requirement. The ratio of 260/280 was actually
used to assess the purity of the DNA and a ratio of approximately 1.8 is considered
as pure DNA. Meanwhile, 260/230 ratio is used as a secondary measure for nucleic
acid purity. The common range of this ratio should be ranging between 2.0 to 2.2 and
it was suppose to be slightly higher than the 260/280 ratio. However, the ratio of
260/230 for sample S7 was not in between the range indicates that the sample shows
the presence of contaminants.
4.5
DNA Sequencing
PCR products of fungi sample S7 were purified and sent with primers NS1
and NS8 to the 1st BASE Laboratories Sdn. Bhd., Malaysia for DNA sequencing.
26
Bioedit version 7.0.5.3 was used to view the sequencing result s generated by the 1 st
BASE Laboratories Sdn. Bhd.
Because of some problems that occured during the sequencing period of
sample S7, only NS1 managed to get sequenced. The size of the sequences obtained
is 937 nucleotides. These are shown in Figure 4.4 and Figure 4.5. As the purification
of PCR product shows the presence of contaminants, the raw result of the sequencing
NS1 can support this statement. Based on figure 4.4, we can see that there is a lot of
letter ‘N’ presence in it. Letter N indicate that there are uncertainties of the
nucleotide present and might be because of the broad peak or a sets of overlaying
peak shown by the chromatogram.
NNNCAANNNGNGNNGTATATGCTTGTNTCNTAGGGAACCTGCGGGGTAGNAAATGCTTGTC
TCCCTTGTTAACCCGCGGAAGTAGGGGAAGTTTGTTTTCCTCGTGGAACCTCCGGAAACGGCG
AAAACCTGTTTCCCGGGAGGGACGCGTTATTAGATAAAAAATCAATGCTCTTTGAGCTCTTTG
ATGATTCATAATAACTTTTCGAATCGCATGGCCTTGTGCTGGCGATGGTTCATTCAAATTTCTG
CCCTATCAACTTTCGATGGTAGGATAGTGGCCTACCATNGTTTCAACGGGTAACGGGGAATAA
GGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGC
GCGCAAATTACCCAATCCCGACACGGGGAGGTAGTGACAATACATAACGATACAGGGCCCTT
TCGGGTCTTGTAATTGGAATGAGTACAATGTAAATACCTTAACGAGGAACAATTGGAGGGCA
AGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTT
AAAAAGCTCGTAGTTGAACTTTGGGCTTGGTTGGCCGGTCCGCCTTTTTGGCGAGTACTGGAC
CCAACCGAGCCTTTCCTTCTGGCTAACCATTCGCCCTTGTGGTGTTTGGCGAACCAGGACTTTT
ACTTTGAAAAAATTAGAGTGTTCAAAGCAGGCCTTTGCTCGAATATATTAGCATGGAATAATA
GAATAGGACGTTATGGTTCTATTTTGTTGGTTTCTAGAACATCNTAATNTTAATAGGAACGGTC
GGGGGCATCAGTATTTCAGTGNAAGANNTGAAATTCTTAAATTACTGAAGACTAATTCTGNN
AAACATTTGCCAGGACGTTTTCTTAATAAANACCAAAGTTAGGGGATCGATNTTATCCG
Figure 4.4
4.6
NS1 sequence for sample S7
Homology Similarity Search by Using Basic Local Alignment Search
Tool (BLAST)
BLAST is a tool used to find out a sequence in database by submitting a
particular sequence and it applies a statistical analysis which is used to compare the
27
significance of each match. The sequence matching can be homologous or related to
the particular sequence and there are many types of BLAST including BLASTp for
amino acids, BLASTn for nucleotides, BLASTx, and other types (Branes, 2007).
BLAST is one of the most important bioinformatics software because it is
effective and fast to identify the unknown sequence data. The software can be
updated and is developed by NCBI (National Center for Biotechnology Information)
(Bedell et al., 2003). In this study, the full sequences of sample S7 were submitted as
query to BLASTn to find homology and similarity after the sequence had been edited
using BIOEDIT software,. The full number of bases viewed was near to full length
of 18S rRNA gene. The scores obtained for sample S7 were shown in the Figure 4.5.
Figure 4.5
Blast result for sample S7.
28
On the BLAST result based on Figure 4.5, we can see that Meyerozyma
guilliermondii (JN546137.1) appear as the first result with the query coverage about
83% and the maximum identical about 97%. However, this result is still uncertain.
To support the result, phylogenetic tree was constructed by using Molecular
Evolutionary Genetic Analysis (MEGA) software.
4.7
Multiple Sequence Alignment
Multiple sequence alignment is a tool that being used to align three or more
biological sequences generally protein, DNA or RNA. In most cases, the set of query
sequence are assumed to have an evolutionary relationship which mean they might
come from the same ancestor. In this study, ClustalX is being used to align all the 10
sequences which one of them is the sequence of sample S7 and the other 9 is the
sequences that have been retrieved from the gene bank.
4.8
Phylogenetic Tree Construction
Phylogenetic tree shows the evolutionary relationship among organisms and
genes which show their comparability and closeness by using the structure of tree
(Salami and Vandamme, 2003).
The most common method used to construct the phylogenetic tree is neighbor
joining method because it is simple and fast. MEGA software is one of the best
software that available for constructing neighbour joining tree (Hall, 2008).
Figures 4.6 show the rooted and scaled phylogenetic trees of sample S7
constructed by using neighbor joining method with 1000 bootstrap replicates.
Saccharomyces cerivisae was used as the out group of the tree.
29
Figure 4.6
Phylogenetic tree of sample S7
From the Figure 4.6 we can see that sample S7 is closely related to
Meyerozyma guilliermondii (JN546137.1) as they were sisters in the tree with
slightly different in terms of their genetic material and come from the same ancestor
which is Pichia guillermondii
30
CHAPTER 5
CONCLUSIONS AND FUTURE WORKS
5.1
Conclusions
In this study, fungi S7 that was isolated at Hutan Rekreasi UTM were
successfully being identified using 18S rRNA molecular approach. The fungal DNA
were isolated by using Wizard Genomic DNA Purification Kit (Promega). The 18S
rRNA genes were being amplified by using polymerase chain reaction (PCR) and
sent for DNA sequencing.
The fungal sequence and phylogenetic tree were assembled and analyzed by
using several bioinformatics software such as ClustalX, Bioedit and MEGA. The
molecular approach using 18S rRNA identification which gave a more reliable result
compared to conventional method which identify the fungi by using microscopic
examination and based on morphology was successfully used in this study. It is also
proven that based on molecular analysis, it can be conluded that sample S7 as
Meyerozyma guillermondii
pollutants (POPs).
which can be used to degrade persistent organic
31
5.2.
Future Works
1. The same method can also be used to identify other fungal samples from
different natural sources like soil, water and clinical samples.
2.
The potential of S7 can also be explored in application study such as
biodegradation toxic dye compounds.
32
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37
APPENDIX A
BLASTn search results of S7
>gb|JN546137.1| Meyerozyma guilliermondii 18S ribosomal RNA gene, partial sequence
Length=1522
Score = 1317 bits (713), Expect = 0.0
Identities = 761/787 (97%), Gaps = 10/787 (1%)
Strand=Plus/Plus
Query 151 TTATTAGATAAAAAATCAATGCTCTTTGAGCTCTTTGATGATTCATAATAACTTTTCGAA 210
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 124 TTATTAGATAAAAAATCAATGCTCTTTGAGCTCTTTGATGATTCATAATAACTTTTCGAA 183
Query 211 TCGCATGGCCTTGTGCTGGCGATGGTTCATTCAAATTTCTGCCCTATCAACTTTCGATGG 270
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 184 TCGCATGGCCTTGTGCTGGCGATGGTTCATTCAAATTTCTGCCCTATCAACTTTCGATGG 243
Query 271 TAGGATAGTGGCCTACCATNGTTTCAACGGGTAACGGGGAATAAGGGTTCGATTCCGGAG 330
||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||
Sbjct 244 TAGGATAGTGGCCTACCATGGTTTCAACGGGTAACGGGGAATAAGGGTTCGATTCCGGAG 303
38
Query 331 AGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAA 390
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 304 AGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAA 363
Query 391 TCCCGACACGGGGAGGTAGTGACAATACATAACGATACAGGGCCCTTTCGGGTCTTGTAA 450
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 364 TCCCGACACGGGGAGGTAGTGACAATACATAACGATACAGGGCCCTTTCGGGTCTTGTAA 423
Query 451 TTGGAATGAGTACAATGTAAATACCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCC 510
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 424 TTGGAATGAGTACAATGTAAATACCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCC 483
Query 511 AGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCT 570
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 484 AGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCT 543
Query 571 CGTAGTTGAACTTTGGGCTTGGTTGGCCGGTCCGCCTTTTTGGCGAGTACTGGACCCAAC 630
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 544 CGTAGTTGAACTTTGGGCTTGGTTGGCCGGTCCGCCTTTTTGGCGAGTACTGGACCCAAC 603
Query 631 CGAGCCTTTCCTTCTGGCTAACCATTCGCCCTTGTGGTGTTTGGCGAACCAGGACTTTTA 690
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 604 CGAGCCTTTCCTTCTGGCTAACCATTCGCCCTTGTGGTGTTTGGCGAACCAGGACTTTTA 663
Query 691 CTTTGAAAAAATTAGAGTGTTCAAAGCAGGCCTTTGCTCGAATATATTAGCATGGAATAA 750
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 664 CTTTGAAAAAATTAGAGTGTTCAAAGCAGGCCTTTGCTCGAATATATTAGCATGGAATAA 723
Query 751 TAGAATAGGACGTTATGGTTCTATTTTGTTGGTTTCTAGAAC-ATCNTAATN-TTAATAG 808
||||||||||||||||||||||||||||||||||||||| || ||| |||| |||||||
Sbjct 724 TAGAATAGGACGTTATGGTTCTATTTTGTTGGTTTCTAGGACCATCGTAATGATTAATAG 783
39
Query 809 GAACGGTCGGGGGCATCAGTATTTCAGT-GNAAGANNTGAAATTCTTAAATT-ACTGAAG 866
| ||||||||||||||||||||| |||| | ||| ||||||||||| ||| |||||||
Sbjct 784 GGACGGTCGGGGGCATCAGTATT-CAGTTGTCAGAGGTGAAATTCTTAGATTTACTGAAG 842
Query 867 ACTAATT-CTGNNAAA-CATTTGCCA-GGACGTTTTC-TTAATAAANACC-AAAGTTAGG 921
||||| | ||| ||| ||||||||| |||||||||| ||||| || | | |||||||||
Sbjct 843 ACTAACTACTGCGAAAGCATTTGCCAAGGACGTTTTCATTAATCAAGAACGAAAGTTAGG 902
Query 922 GGATCGA 928
|||||||
Sbjct 903 GGATCGA 909
40
APPENDIX B
Sequencing result for fungi sample S7 using chromatogram