i Biosorption of Copper by Nepenthes Ampullaria-Associated-Endophytic Fungi by Wong Changi Thesis submitted in partial fulfilment of the requirements for the degree of Master of Science (by research) Faculty of Engineering, Computing and Science Swinburne University of Technology 2015
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i
Biosorption of Copper by Nepenthes
Ampullaria-Associated-Endophytic
Fungi
by Wong Changi
Thesis submitted in partial fulfilment of the requirements for the
degree of
Master of Science (by research)
Faculty of Engineering, Computing and Science
Swinburne University of Technology
2015
ii
Abstract
In recent years, environmental pollution by heavy metals has caused increasing ecological
damage and led to global public health concerns. Biosorption is one of the ways to deal
with heavy metal pollution. In this study, endophytic fungi were (a) isolated from the
carnivorous plant Nepenthes ampullaria (b) assessed for their resistance against heavy
metal copper and (c) evaluated for their biosorption capacity. In total, 147 fungal isolates
were isolated from Nepenthes ampullaria and only 11 (7.5%) of the total isolates were
capable to resist copper concentration up to 1000 ppm. The 11 fungal isolates were
identified through molecular method, and grouped as members of the Phomopsis,
Diaporthe, Nigrospora, and Xylaria. The fungal isolate NA40 related to Xylaria sp.
achieved the highest biosorption capacity of of 73.26 mg/g using live biomass, thus
chosen for study of proteome expression in response to copper. Three different copper
concentrations (0, 300, 500 ppm) were used in the study. Results show that there are 11
protein spots being up-regulated and 1 protein spot down-regulated in response to copper.
The protein spots were identified to be related to the enzymes involved in heat shock
protein, DNA repair and antioxidant reaction. This study on Xylaria serves as a baseline
study for the response of the fungus to copper.
iii
Acknowledgement
“I will give you every place where you set your foot, as I promised Moses. – (Joshua 1:3)”
First and foremost, thanks be to God for the blessing throughout my life and last forever.
A special word of gratitude to my darling, Julia Wee, for all her love and support.
My family, especially my parents and grandparents who provide me with everything I need, love, support and encouragement.
A person who offers his unreserved help and guidance, who I must offer my profoundest
gratitude - my research supervisor, Dr. Moritz Müller. Instead of being like a supervisor,
I feel more like a friend, who is extremely enthusiastic about any kind of research! I am
looking forward for the next round of the researches!
My co-supervisor, Dr. Daniel Tan and Dr. Samuel Lihan, who provided me with help and
guidance throughout the project experiment.
A Big thanks goes to my lab mates and friends, who share and discuss the knowledge of
different fields with me, and also the laboratory officers, who provided me with help and
guidance throughout my bench work period.
Deepest appreciation to all. The simple phrase “thank you” cannot present how much I
feel thankful to you. Without you, this research as well as the dissertation would not have
been possible.
May God bless you and your family, abundantly.
iv
Declaration
I, Wong Changi, hereby declare that this research project entitled “Biosorption of Heavy
Metal (Copper) and Proteomics Study on Nepenthes ampullaria Associated Endophytic
Fungi” is original and contains no material which has been accepted for the award to the
candidate of any other degree or diploma, except where due reference is made in the text
of the examinable outcome; to the best of the candidate’s knowledge contains no material
previously published or written by another person except where due reference is made in
the text of the examinable outcome; and where the work is based on joint research or
publications, discloses the relative contributions of the respective workers or authors.
(WONG CHANGI)
Date: 1st May 2015
v
Publication Arising from this Thesis
The work described in this thesis has been submitted as described in the following:
Wong, C, Tan, D, Lihan, S, Mujahid, A & Müller, M "Biosorption of Copper (Cu) by Endophytic Fungi Isolated from Nepenthes ampullaria", Applied Microbiology and Biotechnology (Manuscript under consideration)
vi
Table of Contents
Page
List of Figures ix
List of Tables xi
1 Introduction 1
1.1 Heavy Metal 1
1.1.1 Copper Pollution 1
1.1.2 Copper Toxicity 5
1.2 Current Technologies for Heavy Metal Removal 7
1.2.1 Chemical Precipitation 7
1.2.2 Ion Exchange 8
1.2.3 Electrodialysis 8
1.2.4 Semiconductor Photocatalysis 9
1.2.5 Membrane Filtration 10
1.2.6 Phytoremediation 11
1.3 Biosorption 13
1.3.1 Biosorbents 15
1.4 Fungal-plant Symbiotic Interaction 17
1.4.1 Endophytic Fungi 17
1.4.2 Heavy Metal Tolerance of Endophytic Fungi 20
1.4.3 Biosorption of Heavy Metal using Endophytic Fungi 21
1.5 Pitcher plants (Nepenthes) as Source of Endophytic Fungi 22
1.5.1 Distribution 23
1.5.2 Habitat 24
1.6 Proteomics - Regulation of Fungi Proteins in Response to
Heavy Metal Stress
25
1.7 Aims of the Present Study and Dissertation Outline 26
The endophytic fungi isolation for the plant samples collected during the scientific
expedition was conducted on site. The isolation of endophytic fungi was done on the same
day (less than 3 hours) as the plants were collected, right after return from the forest, to
ensure the freshness of the plant samples. Stone et al. (2011) suggested the importance of
the endophytic fungi isolation to be carried out as quickly as possible after the collection
of the sample plant, usually within 2 days’ time. The sampling site was remote, had no
electricity, and could only be accessed by small wooden long boats, thereby limiting the
availability of common laboratory equipment. To avoid contamination, the isolation was
carried out using a self-made lamina flow (Figure 2.6). The self-made lamina flow is
made of a plastic box with a plastic paper covered at the mouth of the box. Two small
hand size holes were made on the plastic cover. The inner and outer parts were sterilized
- 33 -
using 75% ethanol and all the autoclaved water (bottles) and autoclaved beakers were
surface wiped using the 75% ethanol before putting into the box. Additionally, control
plates were prepared and potential contaminants removed from the collection (see 2.2.3
for more details on the procedure).
Figure 2.6: A self-made plastic box - I was doing the endophytic fungi isolation at the
site.
2.2.2 Kota Samarahan Roadside
The plants were collected in the morning and stored at 4oC for 3 hours before carrying
out the endophytic fungi isolation. The isolation was carried out in a biosafety cabinet,
located at Swinburne University of Technology, Sarawak.
2.2.3 Endophytic Fungi Isolation
In order to isolate the endophytes, the plant samples have to be surface sterilized to kill
off all the microorganism that live on the surface of the plant such as epiphytic
microorganisms. A few different chemical solution were used for the surface sterilization
of plant samples such as ethanol (Petrini & Dreyfuss 1981), formaldehyde (Kreisel &
- 34 -
Schauer 1987), and sodium hypochlorite solution (Clark et al. 1983). Different surface
sterilization methods with different concentration of the chemical solution and different
surface sterilization timing will yield different endophytic fungi species (Schulz et al.
1993).
The collected plant samples were cut into small pieces (approximately 1cm3) using sterile
surgical blades and surface sterilized using 75% ethanol for 15-30 seconds. After that, the
plant tissue was dipped into autoclaved distilled water to stop the sterilization process and
surface dried by using autoclaved tissue paper. The plant tissue was then placed on Yeast
Extract Glucose Chloramphenicol Agar (YGCA) plates, which contains 1% of the
antibiotic chloramphenicol which will inhibits the growth of bacteria (Kohanski et al.
2010). A control plate was made using the autoclaved distilled water that used for plant
sample dipping. The plates were incubated at ambient temperature (Mentawai plant
samples) and 25oC (Kota Samarahan roadside plant samples). The isolation protocol is
modified from Strobel and Daisy (2003). Please refer to the Figure 2.7 for an overview
in form of a flow chart.
- 35 -
Figure 2.7: An overview in form of the isolation of endophytic fungi.
2.2.4 Endophytic Fungi Purification
After a week of the incubation, endophytic fungi were observed growing out from the
surface sterilized plant tissue, on the agar plates (Figure 2.8). The endophytic fungi were
then isolated out from the agar plate and placed on a fresh Potato Dextrose Agar (PDA)
plate by using autoclaved plastic straw. The isolates were sub-cultured until pure fungal
strains were obtained (Figure 2.9).
Plant samples
Cut into small pieces (Approx. 1 cm)
Surface sterilization with 75% ethanol for 15-30 seconds
Washed with autoclaved distilled water
Surface dried using autoclaved paper towel
Place it on yeast extract glucose chloramphenicol agar (YGCA) plates
Incubated at ambient temperature for mentaiwai plant samples and 25oc for kuching kota samarahan roadside
plant samples
- 36 -
Figure 2.8: Endophytic fungi were observed growing out from the surface sterilized plant
tissue.
Figure 2.9: Purified fungal strains.
- 37 -
2.2.5 Short Term Storage of the Isolated Fungi
The isolated fungi were grown on a PDA plate at 25oC for few days until the fungal
hyphae covered 2/3 of the plate. The fungal plates were then kept at 4oC until further use.
This way of fungal storage only last up to 6 months before the next sub-culturing into
new PDA plates (Nakasone et al. 2004). Please refer to the Figure 2.10 for more details.
Figure 2.10: A schematic view of short term storage of isolated fungi.
2.2.6 Long Term Storage of the Isolated Fungi
The isolated fungi was transferred into universal bottle that contains pure barley grains
(the barley was autoclaved for 3 times to ensure the barley is fully autoclaved- clean).
The fungi were grown in the media and stored at 4oC until all the barley in the bottle have
been covered by the fungi hyphae. This method was recorded to be able to store the fungal
up to 5 years in -20oC for Rhizoctonia solani with less than 1% loss in viability (Webb et
al. 2011). Please refer to the Figure 2.11 for more details.
Figure 2.11: A schematic view of long term storage of isolated fungi.
Autoclave barley grains in a universal bottle for 3 times
Transfer the fungal isolates onto the barley and incubate at 25oC until the fungal hyphae fully covered the barley
Store at 4oC for further use
Fungal isolated grown on Potato Dextrose Agar plate at 25CC until the fungal hyphae covered
2/3 of the plate
Keep the plate at 4oC for further use
- 38 -
2.3 Preliminary Screening of the Resistance Isolated Fungi Against the Heavy Metal Copper
The screening started with the roadside isolates (100 – 1,000 ppm), followed by the
Mentawai isolates (800 – 1,000 ppm). The testing was done using the direct transfer
method instead of adapting method during which the isolates were directly transferred
from a fungal plate (containing no heavy metal copper) to a new media that contains
different concentrations of copper.
The fungal isolates were grown on PDA plate for a week before been transferred to the
testing plates containing different copper concentration. Potato Dextrose Agar plates
containing 100 – 1,000 ppm copper (copper (II) sulfate salt) were prepared. An agar block
of the actively growing fungal hyphae from the PDA plates (containing no heavy metal
copper) was transferred onto the PDA plates that contain copper, by using sterilized
plastic straw. All plates were then incubated at 25oC for a week, all the results were
recorded and the fungal isolates that were able to grow on the PDA plates that contained
1,000 ppm of heavy metal copper concentration were chosen and utilised for copper
biosorption experiments. All the preliminary screening was undertaken in triplicates. The
protocol is modified from Iskandar et al. (2011). See Figure 2.12 for an overview of the
modified method.
A total of eleven (11) isolates were chosen for subsequent biosorption experiments (see
section 2.5) and identified using molecular methods (see below).
- 39 -
Figure 2.12: A schematic overview of preliminary screening of the resistance isolated fungi against the heavy metal copper.
2.4 Molecular Identification of the Chosen (11) Fungal Isolates
Traditional endophytic fungi identification is based on morphological characteristics
which heavily rely on the reproductive structure/ sporulation of the fungi. However, most
endophytic fungi do not produce the reproductive structure/ sporulation (Jones & Pang
2012). Besides that, morphological identification of the fungi requires an extensive
taxonomical knowledge (Gherbawy & Voigt 2010). Therefore, a molecular technique
based on the fungi rDNA sequence, the Internal Transcribed Spacer (ITS) region, is often
used for fungi identification (Arnold 2007). In this research, the chosen (11) fungi were
identified by using the molecular technique.
All the fungi were identified using the molecular technique. The fungal isolates were
cultured in a PDA plate for 3 days, and actively growing mycelia was transferred (using
a sterile toothpick) into 30 μl sterile lysis solution (10mM Tris-HCL, 1 mM EDTA, pH8.0;
Weising et al. 1994) in a 1.5 ml microcentrifuge tube. The tube was then kept in -80oC
overnight. A control tube (contains lysis solution without fungal mycelia) was prepared.
On the next day, the mixture was thawed at room temperature and 1µl of the supernatant
used for Polymerase Chain Reaction. The rest of the crude extract was stored at -20°C
The Fungal Isolates were cultured on Potato Destrose Agar Plate for a Week
Potato Dextrose Agar Plates that contains 100 – 1000 ppm of Heavy Metal Copper were Prepared
An Agar Block of the Actively Growing Fungal Hyphae was transferred onto the Heavy Metal Containing Agar plates
Incubate the Plates at 25oC for a week
- 40 -
until further usage. The fungal DNA extraction protocol is modified from Huhndorf et al.
(2004).
The universal fungal forward and reverse primers, ITS 4 {5’-
TCCTCCGCTTATTGATATGC-3’} and ITS5 {5’-
GGAAGTAAAAGTCGTAACAAGG-3’}, were used in the fungal DNA amplification.
Twenty two (22) μl of the pcr reaction master mix (BIOLINE) were transferred into a
sterile 0.3 ml PCR tube together with 1 μl each of forward and reverse primers and 1 μl
of the genomic DNA. A negative control (PCR mixture with 1 μl supernatant from the
control tube) was prepared.
The Polymerase Chain Reaction (PCR) consisted of an initial denaturing step of 5 minutes
at 94°C followed by 35 cycles (50 seconds at 94°C, 50 seconds at 54°C and 50 seconds
at 72°C), followed by a final extension step at 72°C for 10 minutes. The PCR products
were resolved by electrophoresis through 1% agarose gels in TAE and visualized by
staining with ethidium bromide for 10 minutes and distaining for 15 minutes. There is no
band observed from the control, which indicates the works is clean (Figure 2.13). The
PCR products were then purified and sent for sequencing to the Beijing Genome Institute
(BGI). The sequences obtained were analysed using the National Center for
Biotechnology Information (NCBI - USA) database and a phylogenetic tree was
constructed from genetic distance and bootstrap values calculated using MEGA 6
(Tamura et al. 2013). Please refer to the Figure 2.14 for an overview in form of a flowchart.
- 41 -
Figure 2.13: Polymerase Chain Reaction (PCR) results – gel bands.
- 42 -
Figure 2.14: A schematic overview of molecular identification of the chosen (11) fungal
isolates.
Culture the 11 Fungal Isolates on Potato Destrose Agar Plate for 3 Days
Incubates the tube at -80oC overnight
Transfer a Minimum Amount of Actively Growing Fungal Mycelia into Sterile 30 μl Lysis Solution (TE Buffer) in 1.5 ml Microcentrifuge Tube
The Mixtures Were thawed at Room Temperature and 1 μl of Genomic DNA Solution was transferred into the Polymerase Chain Reaction Mixture
DNA Sequencing at Beijing Genomics Institute
Polymerase Chain Reaction at: Initial Denaturing - 5 mins at 94°C (35 Cycles) Denaturation - 50 seconds at 94°C Annealing - 50 seconds at 54°C Elongation - 50 seconds at 72°C Final Elongation - 72°C for 10 minutes Storage - 4oC until further use
A Phylogenetic Tree Was Constructed From Genetic Distance and Bootstrap Values Calculated Using MEGA 6
The obtained DNA Sequences were Analysed Using the National Center for Biotechnology Information (NCBI - USA) database.
- 43 -
2.5. Evaluation of Biosorption Capacity of the Chosen Fungal Isolates
2.5.1 Heavy Metal Copper Biosorption by Live Fungal Biomass
Potato Dextrose Broth (PDB) supplied with 500 ppm copper (copper (II) sulfate salt) was
prepared. Fungal isolates were grown on PDA plates (containing no heavy metal copper)
for a week before transferal into Potato Dextrose Broth (PDB) containing 500 ppm copper.
Three cylindrical agar plugs of the actively growing fungal hyphae from the PDA plates
were transferred into the PDB containing 500 ppm copper using sterilized plastic straws.
The mixtures were then incubated at 25oC, under static condition for 2 months.
After the 2 months of incubation (Figure 2.15), the fungal biomass were filtered
usingfilter paper (Whatman A1) and dried at 70oC. The weight of the dried fungal biomass
were measured and recorded. The final concentration of heavy metal was measured using
Atomic Absorption Spectrometer (AAS; Xplor AA (Serial No. A6945)). Please refer to
the Figure 2.16 for more details.
The biosorption capability of 1 gram living fungal biomass was calculated using the
following formula (Zafar et al. 2007):
Q [mg/g] = (Ci – Cf [mg/L] / M [g]) V [L]
where Q is mg of metal ion absorbed per gram of fungal biomass [mg/g], Ci and Cf are
the initial and final concentrations of the metal in the solution [mg/L]. M is the amount
of the added (bio)sorbent to the reaction mixture [g] and V is the volume reaction mixture
[L].
- 44 -
Figure 2.15: Fungal isolates were growing in the potato dextrose broth supplied with 500ppm of copper.
- 45 -
Figure 2.16: A schematic overview in form of a flowchart of the heavy metal copper
biosorption by Live fungal biomass.
The 11 Fungal Isolates were cultured on Potato Destrose Agar Plate for a Week
Potato Dextrose Broths that supplied with 500 ppm of Heavy Metal Copper were prepared
An Agar Block of the Actively Growing Fungal Hyphae were Transferred into the Heavy Metal Containing Broth
plates
Separates the Fungal Biomass and the Broths by Filtration using Filter Paper
Fungal Biomass
Dried at 70oC
Measure the Weight of the Dried Biomass
Broth
The Final Concentration of Heavy Metal Copper using were Measured Using Atomic Absorption Spectrometer
The Mixtures were incubated at 25oC for 2 months
- 46 -
2.5.2 Heavy Metal Copper Biosorption by Dead Fungal Biomass
A single cylindrical agar plug of 5 day old fungal cultures was inoculated into 200 ml of
potato dextrose broth (PDB) and incubated for 2 months at 25oC, under static conditions.
After 2 months of incubation, the fungal biomass was filtered using filter paper, dried and
killed at 70oC. The dried biomass was then ground into powder by using pestle and mortar
and the powdered fungal biomass stored in 1.5 ml centrifuge tubes for further use.
Autoclaved distilled water supplied with 500 ppm copper (Copper(II) sulfate salt) was
prepared. The powdered fungal biomass was added into 10 ml of autoclaved distilled
water containing 500 ppm copper, and incubated for 2 months at 25oC. After 2 months of
incubation, the dead fungal biomass was filtered out and the final concentration of the
heavy metal copper was measured using Atomic Absorption Spectrometer (AAS; Xplor
AA (Serial No. A6945)). The protocol is modified from Martínez-Juárez et al. (2012).
Please refer to the Figure 2.17 for an overview in form of a flowchart.
The biosorption capability of 1 gram dead fungal biomass was again calculated as follows
(Zafar et al. 2007):
Q [mg/g] = (Ci – Cf [mg/L] / M [g] ) V [L]
where Q is mg of metal ion absorbed per gram of fungal biomass [mg/g], Ci and Cf are
the initial and final concentrations of the metal in the solution [mg/L]. M is the amount
of the added (bio)sorbent to the reaction mixture [g] and V is the volume reaction mixture
[L].
- 47 -
Figure 2.17: A schematic overview in form of a flowchart of the heavy metal copper biosorption by Dead fungal biomass.
The 11 Fungal Isolates were cultured on Potato Dextrose Broth for a Month
Separates the Fungal Biomass and the Broths by Filtration using Filter Paper
The Final Concentration of Heavy Metal Copper using were Measured Using Atomic Absorption Spectrometer
The Mixtures were incubated at 25oC for 2 months
Fungal Biomass were Harvested
Dried and Killed at 70oC
A known amount of Fungal Dried Biomass were mixed with Autoclaved Distilled water Supplied
with 500ppm of Heavy Metal Copper.
The Fungal Biomass were Powdered Using Pestle and Mortar
- 48 -
2.6 Proteomic Analysis of the Best Fungal Strain (NA40) on Heavy Metal Copper
Fungi are known to produce or overexpress certain enzymes in response to heavy metal
induced oxidative stress, and proteomics provides a way for the stress response to be
studied (Washburn and Yates III 2000; Rabilloud et al. 2005).
Fungal isolate NA40 achieved the best biosorption capacity (live biomass) of heavy metal,
thus it was chosen to perform this analysis. In this study (chapter 4), the isolates NA40
was cultivated in 3 different conditions – potato dextrose broth (PDB) without copper,
PBD with 300 ppm and 500 ppm copper concentration. The chosen fungal isolate NA40
was inoculated into the prepared PDB solutions and incubated at 25oC for 3 weeks, under
static condition. After 3 weeks of incubation, the fungal biomass was stored at 4oC before
it was brought to Agrobiotechnology Institute Malaysia (ABI) to perform the protein
extraction and analysis.
Please refer to the Figure 2.18 for an overview in form of a flowchart and to chapter 4 for
more details.
Figure 2.18: An overview in form of a flowchart of the fungal incubation in PDB with three different concentration of heavy metal copper concentration.
The Mixture were incubated for 3 weeks at 25oC under static condition
The Fungal Biomass were stored at 4oC before bringing to Agrobiotechnology Institute for
Protein Analysis
The Fungal Isolate (NA40) were Cultured in Potato Dextrose Broth supplied with 0, 300 and
500 ppm of Heavy Metal Copper.
- 49 -
2.6.1 Fungal Proteome Preparation
The fungal biomass was harvested by spinning down using a temperature controlled
centrifuge at 10,000 g at 4oC, for 10 minutes. After that the supernatants were discarded
and the fungal biomass rinsed with deionised water and again span down using a
temperature controlled centrifuge at 10,000 g at 4oC, for 10 minutes. This rinsing step
was repeated for 2 times. The fungal biomass was grounded into fine powder using mortar
and pestle in the presence of liquid nitrogen. TCA-acetone extraction was performed by
mixing each of the 1g of the powdered fungal biomass with 1.8 ml of 10% trichloroacetic
acid in cold acetone containing 0.07% β-mercaptoethanol and vortex at the temperature
of 4oC. After that, the mixture was incubated in 20oC overnight. On the next day, the
mixture was centrifuged at 10,000 g at 4oC for 15 minutes. The supernatant was discarded
and the pellet was re-suspended in rising solution (each of the 1 g with 1.8 ml of rinsing
solution) that contains 0.07% β-mercaptoethanol in cold acetone, which was then
incubated at -20°C for 1 h (mixed every 15 min intervals) and re-centrifuged at 10,000g
at 4oC for 15 minutes. The supernatant was discarded and this rising steps was repeated
for 2 times. The pellet was then vacuum-dried and re-suspended with lysis buffer and
stored at -80oC. The protocol is modified base on the paper written by Pavoković et al.
(2012). Please refer to the Figure 2.19 for more details.
- 50 -
Figure 2.19: An overview in form of a flowchart of the fungal proteome preparation.
Vortexed the Mixtures at 4oC for 10 minutes
One (1) gram of the Powdered Fungal Biomass was Transferred into 1.5ml Microcentrifuge Tube
The Fungal Biomass were Transferred into clean 50 ml falcon tube
The supernatant were discarded
The amount of 1.8 ml of of 10% Trichloroacetic Acid in Cold Acetone Containing 0.07% β-mercaptoethanol was added to each of the tubes
Powered the Fungal Biomass by Using Mortar and Pestle with Liquid Nitrogen
Incubated at 20oC Overnight
Spin down the Fungal Biomass at 10,000g at 4oC
Centrifuged at 10000 g at 4oC for 15 minutes
Twenty Five (25) ml of Deionised Water were added into the tubes
Spin down the Fungal Biomass using using Temperature controlled Centrifuge at 10,000g at 4oC
The Supernatant were Discarded
The Supernatant were Discarded
Resuspened with Lysis Buffer
The pellets were vacuum-dried
The pellet was Resuspended in Rinsing Solution
Incubated at 4oC for 1 hour (mixed every 15 min interval)
Centrifuged at 10,000g at 4oC for 15 minutes
The Supernatant were Discarded
Stored at -80oC for Further Usage
Rep
eate
d Tw
ice
Rep
eate
d Tw
ice
- 51 -
2.6.2 Total Protein Measurement by Bradford Assay
Bradford Assay, introduced by Bradford in the year of 1976, is a protein determination
method that has been used widely for determination of protein concentration. All the
proteins extracted from fungal biomass, collected from the three different solutions, were
analysed for their total protein concentration using the Bradford Assay kit (Biorad
Bradford Reagent assay). The total concentrations of the extracted proteins were
measured and recorded.
2.6.3 Two-dimensional Gel Electrophoresis (2-DE)
Two-dimensional gel electrophoresis (2-DE) is a gel-based proteomics technique that
have been widely used for the separation, detection and analysis of proteome from
complex biological sources which was 1st introduced by O'Farrell (1975). By using this
technique, proteins are separated based on the different properties. During the 1st
dimension, protein will be separated base on their different isoelectic point in the gel
matrix by Isoelectric focusing (IEF). The separated protein will then be re-separated again
in second dimension, based on their different molecular weight, in Polyacrylamide gel by
sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE).
2D gels of the control and each treatment were run in triplicates. Isoelectric focusing (IEF)
was performed using 13 cm Nonlinear IPG-strips (pH range 3-10). The IPG-strips were
initially rehydrated for 12 hours in the presence of 70 μg of protein. IEF was performed
using Biorad Protean i12 with standard protocol based on Biorad Handbook (IEF
Protocol), at 20 °C in a stepwise manner: 500 V (2 h), 1.0 kV (1 h), 8.0 kV (1 h), 8.0 kV
(28000 VhS) and finally 750 V (hold). Please refer to Figure 2.20 for more details. The
strips were equilibrated in equilibration buffer (based on GE Healthcare 2D SDS PAGE
Handbook) containing 50 mM Tris–HCl pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v)
SDS, 0.002% (w/v) bromophenol blue and 1% (w/v) dithiothreitol (DTT) for 15 minutes,
followed by equilibrated in the same equilibration buffer containing 2.5% (w/v)
iodoacetamide instead of DTT for another 15 minutes. The second dimension separation
was performed in 12% polyacrylamide gels, at 20oC, using SE 600 Ruby system (Hoefer
SE 600 Ruby (Amersham Biosciences)), with the running buffer contains 25 mM Tris–
HCl, 192 mM glycine, 0.1% (w/v) SDS, at 10mA/gel (15min) and 20mA/ gel (3h 30min).
- 52 -
The gels were stained with silver staining (Shevchenko et al., 1996; see section 2.6.4).
Please refer to Figure 2.21 for more details.
Figure 2.20: An overview in form of a flowchart of Isoelectric focusing.
The mixture was pipetted into the rehydration tray as a streak slightly shorter than the strip to be rehydrated (Bubble formation was prevented)
The IPG-strip was allowed to rehydrated for 12 hours and IEF for 4-5 hours
Rehydration and IEF parameter were set
IPG-strip was placed into a slot with the dried gel side down
Protective film was removed from the IPG-stips from the acidic (+) end
Approx. 1 ml of cover fluid (mineral oil) were overlaid onto the strip
The Protein Samples was mixed with rehydration buffer to make the total volume of 200 μl with 70 μg protein concentration
- 53 -
Figure 2.21: An overview in form of a flowchart of two-dimensional gel electrophoresis (2-DE).
Equilibrated the strips with SDS equilibration 1 solution (DTT) for 15 minutes
Rinsed the IPG-strips with running buffer
Poured away the SDS equilibration 2 solution
Placed the IPG-strips into the strip holders
Rinsed the IPG-strips with running buffer
Poured away the SDS equilibration 1 solution
Equilibrated the strips with SDS equilibration 2 solution (IAA) for 15 minutes
IPG-strips removed from the slots
Inserted the strips into the gel
Inserted the protein marker
Overlay the strips with agarose sealing solution
Transferred the glass plate into the tank
The gels were allowed to run for 3 hours 45 minutes with the set parameter
- 54 -
2.6.4 Silver staining for SDS-PAGE
The silver staining protocol was modified from Shevchenko et al., (1996). The gel was
fixed with fixation solution for 30 minutes, followed by 30 minutes in sensitizing solution.
After that the gel was rinsed with Millipore water for 5 mins (3 times). After rinsing, the
gel was submerged in silver nitrate solution for 20 minutes. After the incubation, the silver
nitrate was discarded and staining solution was added onto the gel. The gel was incubated
in the staining solution until the protein spots were started to appear (5-10 minutes). The
staining solution was then discarded and stopping solution was added onto the gel to stop
the staining process. The gel was leaved in the stopping solution for 10 minutes and rinsed
with Millipore water for 3 times. After rinsing the gel was stored in conserving solution
until the further use. Please refer to Figure 2.22 for more details and Figure 2.23 for the
gel image after silver staining.
Figure 2.22: An overview in form of a flowchart of silver staining for SDS-PAGE.
Fixed the gel with the fixation solution for 30 minutes
Rinsed the gel using Millipore water for 5 minutes
Rinsed with Millipore water for 10 minutes
Sensitized the gel with the sensitizing solution for 30 minutes
Submerged in silver nitrate solution for 20 minutes
Stained the gel using staining solution unti the protein spots were started to appear (5-10 minutes)
Stopped the staining using stopping solution (incubated for 10 minutes)
Rep
eate
d Th
ree
times
Conserved the gel in conserving solution until further use
- 55 -
Figure 2.23: Silver stained gel image taken using Cannon digital camera, at the bench of
the lab.
- 56 -
2.6.5 Protein Identification and Database Search
Stained gels were digitized by using image Scanner (GS800 Desitometer (Biorad)) and
the protein spots analysis were performed using Progenesis Samespots samespot software,
with the Max fold change ≥ 2, and Anova p-value ≤ 0.05. Figure 2.24, 2.25 and 2.26
show the images scanned using the scanner, with blue spots indicates the protein spots of
interest. The chosen proteins spots were then manually excised from the stained 2D gels
and destained followed by in gel digestion using trypsin overnight at 25oC (Shevchenko
et al. 2007). The peptides were extracted from the gel pieces by using 50% of acetonitrile
and 100% acetonitrile for the second time extraction. The solution is then vaccum dried
and stored for further identification. Please refer to Figure 2.27 for more details.
Protein identification were done by Norasfaliza Rahmad which was accomplished by
mass spectrometry. Peptide Mass Fingerprinting (PMF) data search was performed using
Swiss-Prot database. The obtained protein ID is then further analysed and studied.
- 57 -
Figure 2.24: Protein spots produced by the fungal isolate NA40 in the PDB with no heavy
metal copper, gel image was taken using image Scanner (GS800 Desitometer (Biorad)).
- 58 -
Figure 2.25: Protein spots produced by the fungal isolate NA40 in the PDB supplied with
300ppm of heavy metal copper, gel image was taken using image Scanner (GS800
Desitometer (Biorad)).
Figure 2.26: Protein spots produced by the fungal isolate NA40 in the PDB supplied with
500ppm of heavy metal copper, gel image was taken using image Scanner (GS800
Desitometer (Biorad)).
- 59 -
Figure 2.27: An overview in form of the in-gel digestion of the protein spots.
Cut the gel spot into small pieces (1-2 mm)
A volume of 50 μl of 100% ACN was added into the tube
A volume of 150 μl of 100 mM NA4(HCO3) was added into the tube
Washed the gel with solution (50% ACN in 100 mM of NA4(HCO3)) for 20 minutes
Removed the solution
Incubated the tube for 20 minutes in the dark
Placed the gel pieces into 1.5 ml microcentrifuge tube
Washed the gel for 10 minutes
Removed the NA4(HCO3)
Alkylated the protein by adding 150 μl of 55 mM IAA in 100 mM of NA4(HCO3)
Incubated the tube for 15 minutes at room temperature
The gel was dried by speed vacuum for 15 minutes
A volume of 25 μl of 7 ng/μl trypsin solution was added into the tube
A volume of 50 μl of 100% ACN was added into the tube (second extraction)
Collected the solution into a new 1.5 ml microcentrifuge tube
Incubated the tube in the waterbath at 25oC overnight
Removed the tube from waterbath
A volume of 25 μl of 50% ACN was added into the tube
Incubated the tube for 15 minutes
Collected the solution into the 1.5 ml microcentrifuge tube
Stored at -80oC before proceed to protein identification
Dried the solution by speed vacuum
Rep
eate
d Th
ree
times
R
epea
ted
Twic
e
- 60 -
Chapter 3
Biosorption of Copper (Cu) by Endophytic
Fungi Isolated from Nepenthes ampullaria
Changi Wong1*, Daniel Tan1, Samuel Lihan2, Aazani Mujahid2, and Moritz Müller1
1 Faculty of Engineering, Computing and Science, Swinburne University of Technology
Sarawak, 93350 Kuching, Malaysia.
2 Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 93400
Biosorption uses biological materials such as bacteria, algae, yeast and fungi (Volesky
1986) to accumulate heavy metals from wastewater through physico-chemical or
metabolically mediated pathways of uptake (Fourest & Roux 1992). There are several
advantages compared to other approaches such as high efficiency, cost effectiveness, the
possibility of recovering the metal of interest and regeneration of the biosorbent
(Kratochvil & Volesky 1998). Recent studies have shown the capability of using
endophytic fungi as biosorbent to bioabsorb or to remove heavy metal. The endophytic
Microsphaeropsis sp. LSE10 isolated from Solanum nigrum L. plant is capable to biosorb
heavy metal cadmium (Xiao et al. 2010), and another endophytic Mucor sp. CBRF59
isolated from Brassica chinensis plant collected from metal-contaminated soil is able to
biosorb heavy metal cadmium and lead by using its live and dead biomass (Deng et al.
2011).
In this study, endophytic fungi were (a) isolated from of the carnivorous plant Nepenthes
ampullaria (collected from undisturbed and anthropogenically affected areas; Mentawai
Jungle and Kota Samarahan roadside, Kuching); (b) assessed for their resistance against
the heavy metal copper; and (c) their biosorption capacity (live and dead biomass)
evaluated.
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3.2 Methodology
3.2.1 Endophyte Isolation and Purification
Nepenthes ampullaria were collected in the Mentawai Jungle (Miri, Sarawak, Malaysia)
during the Heart of Borneo Initiative; Mentawai expedition 2013, and from roadsides at
Kota Samarahan (Kuching, Sarawak, Malaysia). Isolation of endophytic fungi followed
the modified procedures from Strobel and Daisy (2003). In summary, different parts of
the plants (leaves, pitcher, and roots) were cut into small pieces of 1 cm2 in size and
surface-sterilized by immersion in 70% ethanol for 5 - 15 seconds. After that, the sample
was immersed in sterile distilled water (twice) to stop the sterilization. The sample was
then dried using a sterile cotton cloth and placed on a Yeast Extract Glucose
Chloramphenicol Agar (YGCA) plate, which contains chloramphenicol to suppress the
growth of bacteria. One millilitre of the sterile distilled water that was used to clean the
sample was taken out and poured on another YCGA plate as negative control. The agar
dish was then sealed with parafilm, labelled and incubated at 25°C for 7 days. The growth
of hyphae was observed after 7 days and all of the hyphae were isolated using sterile
plastic straws and placed on fresh YCGA dishes. This step was repeated until pure fungal
colonies were obtained. Purified fungi were cultured on Potato Dextrose Agar (PDA) and
incubated at 25°C.
3.2.2 Preliminary Screening of Heavy Metal Copper Tolerance Fungi
Preliminary screening followed procedures modified from Iskandar et al. (2011). In
summary, a single cylindrical block (agar plug) of 5 day old fungal cultures were placed
on Potato Dextrose Agar plates supplemented with Cu (100 – 1000 ppm), and incubated
at 25oC for 7 days. Growth of the fungal isolates was observed and recorded after the 3rd
and 7th day.
3.2.3 Molecular Identification
Identification of endophytic fungi followed the procedures modified from Huhndorf et al.
(2004). In summary, a small amount of 3 days old mycelia was transferred into sterile 30
μl lysis solution (TE buffer – 10 mM Tris-HCL, 1 mM EDTA, at pH 8) in 1.5 ml
microcentrifuge tubes using a sterile toothpick, and incubated at -80°C overnight. On the
- 64 -
next day, the mixture was thawed at room temperature and 1 µl of the supernatant used
for Polymerase Chain Reaction. The rest of the crude extract was stored at -20°C until
further usage.
Twenty two (22) μl of the master mix (BIOLINE) were transferred into a sterile 0.3 ml
PCR tube together with 1 μl of the each forward and reversed primers (ITS4 {5’-
TCCTCCGCTTATTGATATGC-3’} and ITS5 {5’-
GGAAGTAAAAGTCGTAACAAGG-3’}), and 1 μl of the genomic DNA. The mixture
was then used for polymerase chain reaction.
The Polymerase Chain Reaction (PCR) consisted of an initial denaturing step of 5 minutes
at 94°C followed by 35 cycles (XY seconds at 94°C, 50 seconds at 54°C and 50 seconds
at 72°C), followed by a final extension step at 72°C for 10 minutes. The PCR products
were resolved by electrophoresis through 1% agarose gels in TAE and visualized by
staining with ethidium bromide for 10 minutes and destaining for 15 minutes. The PCR
products were then purified and sent for sequencing. The sequences obtained were
analyzed against the NCBI (USA) database (Zhang et al. 2000) and a phylogenetic tree
was constructed from genetic distance and bootstrap values calculated using MEGA 6
(see Figure 3.1; Tamura et al. 2013).
3.2.4 Biosorption of Copper by Living Fungal Biomass
Biosorption capacity was calculated following procedures outlined by (Zafar et al. 2007).
In summary, three single cylindrical blocks (agar plugs) of 5 day old fungal cultures were
inoculated into 80 ml of potato dextrose broth (PDB), with 500 ppm of heavy metal
copper added, and incubated for 2 months at 25oC, under static conditions.
After 2 months of incubation, the fungal biomass was filtered and dried at 70oC. The
weight of the dried fungal biomass were measured and recorded. The final concentration
of heavy metal was measured using Atomic Absorption Spectrometer (AAS; Xplor AA
- 65 -
(Serial No. A6945)). The biosorption capability of 1 gram living fungal biomass was
calculated using the formula:
Q [mg/g] = (Ci – Cf [mg/L] / M [g]) V [L]
where Q is mg of metal ion absorbed per gram of fungal biomass [mg/g], Ci and Cf are
the initial and final concentrations of the metal in the solution [mg/L]. M is the amount
of the added (bio)sorbent to the reaction mixture [g] and V is the volume reaction mixture
[L].
3.2.5 Biosorption of Copper by Dead Fungal Biomass
Biosorption capacity was calculated following procedures outlined by (Zafar et al. 2007).
A single cylindrical block (agar plug) of 5 days old fungal cultures was inoculated into
200 ml of potato dextrose broth (PDB) and incubated for 2 months at 25oC, under static
conditions. After 2 months of incubation, the fungal biomass was filtered, dried and killed
at 70oC. The dried biomass were then grinded into powder by using pestle and mortar.
The powered fungal biomass were stored into 1.5ml of centrifuge tube for further use.
An small amount of dried fungal biomass were pre-weighted and recorded, and added
into 10 ml of distilled water, with 500 ppm of heavy metal copper added, and incubated
for 2 months at 25oC.
After 2 months of incubation, the dead fungal biomass was filtered out and Cu
concentrations measured using Atomic Absorption Spectrometer (machine model). The
biosorption capability of 1 gram dead fungal biomass was calculated as follows:
Q [mg/g] = (Ci – Cf [mg/L] / M [g]) V [L]
where Q is mg of metal ion absorbed per gram of fungal biomass [mg/g], Ci and Cf are
the initial and final concentrations of the metal in the solution [mg/L]. M is the amount
of the added (bio)sorbent to the reaction mixture [g] and V is the volume reaction mixture
[L].
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3.3 Results and Discussion
Endophyte Isolation and Purification
A Total of 147 fungal isolates were isolated from Nepenthes ampullaria. Ninety two (92)
isolates were from plants collected in Mentawai Jungle, while the other fifty five (55)
isolates were from plants collected at the roadside of Kota Samarahan, Kuching, Sarawak,
Malaysia.
Preliminary Screening of Copper Tolerance among Fungi
Of the 147 fungal isolates, Ninety two (92) isolates were from plants collected in
Mentawai Jungle, while the other fifty five (55) isolates were from plants collected at the
roadside of Kota Samarahan, Kuching, Sarawak, Malaysia. Only 11 managed to survive
in copper concentrations up to 1000 ppm (Table 3.1). Nine out of these 11 isolates (NA8,
NA25, NA27, NA28, NA31, NA40, NA41, S1 and S2) were isolated from a plant
collected from the roadside of Kota Samarahan, while only 2 isolates from Mentawai
Jungle (MNA3 and MNA27) were able to survive at 1000 ppm Cu. These 11 isolates
were chosen to carry out the Heavy Metal (copper) Biosorption Assay by using the living
and dead fungal biomass.
The results clearly showed that fungal isolates isolated from roadside plants display much
higher resistance towards Cu. This can be explained by increased exposure to Cu along
roadsides (and necessary adaptation to survive), as compared to the undisturbed jungle
environment. Zehetner et al. (2009) showed that roadside environments are often
contaminated by automobile traffic with a wide range of contaminants such as heavy
metals, which can be found in the wall of fuel tanks, engines, tires, brake pads and road
surface materials. Han et al. (2014) conducted a research on heavy metal concentrations
in road dust in Kuala Lumpur, and Cu ranked 2nd in concentration among the heavy metal
contaminants. These studies support our findings and indicate that the Nepenthes and their
endophytic fungi along the roadsides of Kota Samarahan have been exposed to heavy
metals released by the automobile traffic and have therefore developed stronger resistance
towards the metals.
- 67 -
Table 3.1: Fungal isolates that manage to survive up to 1000ppm of heavy metal copper
concentration.
Fungal Isolates Nepenthes ampullaria Plant Origin
NA8 Kota Samarahan Roadside NA25 Kota Samarahan Roadside NA27 Kota Samarahan Roadside NA28 Kota Samarahan Roadside NA31 Kota Samarahan Roadside NA40 Kota Samarahan Roadside NA41 Kota Samarahan Roadside
S1 Kota Samarahan Roadside S2 Kota Samarahan Roadside
MNA3 Mentawai Jungle
MNA27 Mentawai Jungle
Molecular Identification of the Top 11 Fungal Isolates
Fungi morphological characterization has been used widely for the identification of fungi.
However, fungi identification by visual examination is rather time consuming, erroneous,
difficult, and requires an extensive taxonomical knowledge, compared to the molecular
technique, which is more sensitive, specific and accurate, and does not demand the
Table 4.3 Down-regulated protein in response to copper.
Spot
Number Protein name Accession number
Treatment/Contro
l (ratio)
300pp
m
500pp
m
33 Cell division protein SepF SEPF_MYCLB 0.20 0.54
- 90 -
Figure 4.1: Up-regulated protein spots in response to copper.
Figure 4.2: Down-regulated protein spot in response to copper.
- 91 -
4.0 Conclusion
This study is the first to perform a proteomic analysis of the fungus Xylaria sp. in response
to heavy metal copper oxidative stress. The analysis shows that the particular fungus is
capable of producing a wide range of enzymes involved in repair of damaged DNA,
antioxidant catalysation, and heat shock proteins. The results can serve as a baseline study
for this particular fungus genus, Xylaria, on heavy metal copper proteome study.
- 92 -
Chapter 5
Summary, Conclusion and Future Work
5.1 Summary
This research study has presented (i) the capability of the isolated endophytic fungi from
Nepenthes ampullaria plants collected from undisturbed and anthropogenically affected
areas (Mentawai Jungle and Kota Samarahan roadside, Kuching) (a) to resist heavy metal
copper and (b) to biosorp copper from solution by using Live and Dead biomass, and (ii)
express different proteins (fungal isolate NA40) in response to copper stress.
In this study, ninety two (92) fungal isolates were isolated from the Nepenthes ampullaria
plants collected in Mentawai Jungle and fifty five (55) fungal isolates from the roadside
of Kota Samarahan Kuching, Sarawak, Malaysia. In total, there were 147 fungal isolates
collected from Nepenthes ampullaria plants, with the capability of the 7.5% fungal
isolates able to resist heavy metal copper concentration up to 1,000 ppm. The highest Cu
biosorption capacity of live biomass was achieved by fungal isolate NA40 (related to
Xylaria sp.; 73.26 mg/g), whereas NA41 (related to Phomopsis sp.) showed to have the
highest Cu biosorption capacity using its dead biomass (73.26 mg/g). To our knowledge,
this is the first reported study on the copper tolerance of Xylaria, Diaporthe and
Nigrospora sphaerica, and copper biosorption using live biomass of Xylaria and
Nigrospora sphaerica and dead biomass of Xylaria, Diaporthe and Nigrospora oryza.
This study highlights that fungal biosorption capacity is highly dependent on the sampling
area (roadside vs. jungle) and the fungal species. Moreover, the results also highlighted
that the different biosorption mechanisms (live- metabolic dependent and dead biomass-
metabolic independent) result in different amounts of copper being removed from the
solutions.
The proteomics analysis of the fungal isolates NA40 (related to Xylaria sp.) showed that
the particular fungus is able to produce a wide range of enzymes to protect itself from the
oxidative stress caused by copper. The proteins produced include enzymes that repair
damaged DNA, antioxidant and heat shock proteins. To our knowledge, this is the first
proteomic analysis study performed on the fungus Xylaria sp. in response to heavy metal
- 93 -
copper oxidative stress and the results obtained can serve as a baseline study for the
particular fungus genus, Xylaria, on heavy metal copper proteome study.
5.2 Future work
There is no doubt that the biosorption capabilities of the eleven fungi are highly promising,
and have the potential to be used as a new biosorbent materials in the near futures. We
have only just begun to ‘scratch the surface' of bioremediation using endophytes and the
biodiversity treasures of Borneo will surely yield many more surprises. However, future
work should expand our current knowledge and involve researches from chemistry,
biochemistry, genetics, and polymer sciences, in order to fully explore the potential of
endophytes in metal removal and (or) recovery.
Other than heavy metal resistant, biosorption and proteomics studies, endophytic fungi
are also known for their antimicrobial (Phongpaichit et al. 2006) and enzymatic properties
(Sculz et al. 2002). The world's first billion-dollar anticancer drug, Paclitaxel (taxol), was
found to be produced by a wide range of endophytic fungi (Strobel & Daisy 2003).
In the present study, a total number of 147 fungal isolates were collected from the
Nepenthes ampullaria plant and we would suggest to use the isolated fungal to carry out
further studies such as antimicrobial and enzymatic testing, to find out/ unleash/
understand more of their hidden abilities. Besides that, we would also suggest to carry
out the tolerance testing and biosorption on other heavy metals such as chromium, lead,
zinc, mercury and uranium. Moreover, pre-treatment of the fungal dead biomass that
might maximise the biosorption capacities, could also be carried out. Last but not least,
de novo (peptide) sequencing study can be used to confirm and expand upon the results
obtained from database searches (Cagney & Emili 2002).
- 94 -
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