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

2 Methodology 28

2.1 Sampling Sites 28

2.1.1 Ulu Mentawai 30

2.1.2 Kota Samarahan Roadside 31

2.2 Nepenthes ampullaria Associated Endophytic Fungi Isolation 32

2.2.1 Ulu Mentawai 32

vii

2.2.2 Kota Samarahan Roadside 33

2.2.3 Endophytic Fungi Isolation 33

2.2.4 Endophytic Fungi Purification 35

2.2.5 Short Term Storage of the Isolated Fungi 37

2.2.6 Long Term Storage of the Isolated Fungi 37

2.3 Preliminary Screening of the Resistance Isolated Fungi Against

the Heavy Metal Copper

38

2.4 Molecular Identification of the Chosen (11) Fungal Isolates 39

2.5 Evaluation of Biosorption Capacity of the Chosen Fungal

Isolates

43

2.5.1 Heavy Metal Copper Biosorption by Live Fungal

Biomass

43

2.5.2 Heavy Metal Copper Biosorption by Dead Fungal

Biomass

46

2.6 Proteomic Analysis of the Best Fungal Strain (NA40) on Heavy

Metal Copper

48

2.6.1 Fungal Proteome Preparation 49

2.6.2 Total Protein Measurement by Bradford Assay 51

2.6.3 Two-dimensional Gel Electrophoresis (2-DE) 51

2.6.4 Silver staining for SDS-PAGE 54

2.6.5 Protein Identification and Database Search 56

3 Biosorption of Copper (Cu) by Endophytic Fungi Isolated from

Nepenthes ampullaria

60

3.1 Introduction 61

3.2 Methodology 63

3.2.1 Endophyte Isolation and Purification 63

3.2.2 Preliminary Screening of Heavy Metal Copper

Tolerance Fungi

63

3.2.3 Molecular Identification 63

3.2.4 Biosorption of Copper by Living Fungal Biomass 64

3.2.5 Biosorption of Copper by Dead Fungal Biomass 65

3.3 Results and Discussion 66

3.4 Conclusion 76

viii

4 Proteomics analysis of the Nepenthes ampullaria associated

endophytic fungus, Xylaria sp.

78

4.1 Introduction 79

4.2 Methodology 81

4.2.1 Culture Conditions 81

4.2.2 Protein Extraction 81

4.2.3 2-DE and Image Analysis of Protein Spots 81

4.2.4 Protein Identification and Database Search 82

4.3 Results and Discussion 83

4.4 Conclusion 91

5 Summary, Conclusion and Future Work 92

5.1 Summary 92

5.2 Future Work 93

References 94

ix

List of Figures

Figure Page

1.1 Basic structure of Nepenthes pitcher. 22

1.2 Distribution map of Nepenthes sp., taken from Carnivorous Plants /

Insectivorous Plants in the Wilderness.

24

2.1 Ulu Mentawai (sampling sites), located at northern part of Gunung Mulu

National Park, indicated by red point (Source: Google Map).

29

2.2 Kuching Kota Samarahan roadsite (sampling sites), indicated by red

point (Source: Google Map).

29

2.3 Nepenthes ampullaria in situ, photographed on site (Mentawai jungle). 30

2.4 Author collecting plant samples collecting at in Mentawai jungle. 31

2.5 Kuching Kota Samarahan Roadside, the area where the Nepenthes

ampullaria was collected.

32

2.6 A self-made plastic box - I was doing the endophytic fungi isolation at

the site.

33

2.7 An overview in form of the isolation of endophytic fungi. 35

2.8 Endophytic fungi were observed growing out from the surface sterilized

plant tissue.

36

2.9 Purified fungal strains. 36

2.10 A schematic view of short term storage of isolated fungi. 37

2.11 A schematic view of long term storage of isolated fungi. 37

2.12 A schematic overview of preliminary screening of the resistance isolated

fungi against the heavy metal copper.

39

2.13 Polymerase Chain Reaction (PCR) results – gel bands. 41

2.14 A schematic overview of molecular identification of the chosen (11)

fungal isolates.

42

2.15 Fungal isolates were growing in the potato dextrose broth supplied with

500ppm of copper.

44

2.16 A schematic overview in form of a flowchart of the heavy metal copper

biosorption by Live fungal biomass.

45

2.17 A schematic overview in form of a flowchart of the heavy metal copper

biosorption by Dead fungal biomass.

47

x

2.18 An overview in form of a flowchart of the fungal incubation in PDB

with three different concentration of heavy metal copper concentration.

48

2.19 An overview in form of a flowchart of the fungal proteome preparation. 50

2.20 An overview in form of a flowchart of Isoelectric focusing. 52

2.21 An overview in form of a flowchart of two-dimensional gel

electrophoresis (2-DE).

53

2.22 An overview in form of a flowchart of silver staining for SDS-PAGE. 54

2.23 Silver stained gel image taken using Cannon digital camera, at the bench

of the lab.

55

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)).

57

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)).

58

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)).

58

2.27 An overview in form of the in-gel digestion of the protein spots. 59

3.1 ITS gene-based phylogenetic tree representing fungal sequences

conserved within the internal transcribed spacer region. The

phylogenetic tree was constructed using Mega 6 with distance method

and sequence distances were calculated using maximum likelihood

method. Boot strap values of 2000 are shown and the cut off value of

50%.

70

4.1 Up-regulated protein spots in response to copper. 90

4.2 Down-regulated protein spot in response to copper. 90

xi

List of Tables

Table Page

1.1 Acid mine drainage and some other industrial activities impact towards

the environment and human.

3

1.2 Malaysia local heavy metal contaminated area and the source of pollution. 4

1.3 Effects of acute and chronic copper poisoning on human organ. 6

1.4 Overview of advantages and disadvantages of each of the five

phytoremediation techniques /approaches.

13

1.5 Overview of biosorbents and their uptake efficiencies for selected metals. 15

1.6 Overview of endophytic fungi and their host plant interaction. 18

1.7 Overview of endophytic fungi and their resistance against heavy metal. 20

1.8 Overview of endophytic fungi and their capability of biosorpt heavy

metal.

21

2.1 GPS coordination for the Nepenthes ampullaria plant samples collected. 28

3.1 Fungal isolates that manage to survive up to 1000ppm of heavy metal

copper concentration.

67

3.2 ITS phylogenetic results for the 11 isolated fungi strains. 69

3.3 Biosorption capacity (mg/g) of the Live biomass of the isolated fungal on

heavy metal copper biosorption.

72

3.4 Biosorption capacity (mg/g) of the Dead biomass of the isolated fungal on

heavy metal copper biosorption.

74

4.1 List of identified proteins produced (upregulated and downregulated) in

response of heavy metal copper.

88

4.2 Up-regulated proteins in response to copper. 89

4.3 Down-regulated protein in response to copper. 89

- 1 -

Chapter 1

Introduction

1.1 Heavy Metal

In recent years, environmental pollution by heavy metals has caused increasing ecological

damage and led to global public health concerns (Tchounwou et al. 2012). Heavy metals

are naturally occurring elements that can be found throughout the earth’s crust and can

be considered as trace elements when present at trace concentrations (mgkg-1 or less) in

agro-ecosystems (He et al. 2005). However, most of the heavy metal have been released

into our environment from anthropogenic activities such as smelting operations, industrial

production, agricultural, mining activities, sewage treatment plant, and domestic garbage

dumps (Yunus et al. 2011). These activities result in increasing concentrations of heavy

metals in our environment and lead to heavy metal pollution (Tchounwou et al. 2012).

Heavy metals are catergorised as severe pollutants due to their toxicity, bioaccumulation

and persistent properties (Tam & Wong 2000). They are neither chemically nor

biologically degraded, and therefore, the pollutants may persist in the environment for

long period of time. For instance, mangrove forest of Tanjung Lumpur are known to be

polluted by several heavy metal such as lead, copper and Manganese due to the

terigeneous and antropogenic activities. The copper and lead are know to be most concern

metals due to their accumulation in aquatic organism consumed by humans (Luoma 1990;

MacFarlane & Burchett 2000).

1.1.1 Copper Pollution

According to the Agency of Toxic Substances and Disease Registry (ATSDR 2004),

approximately 640,000,000 kg of the heavy metal copper (Cu) were released into the

environment by industries in the year of 2000. Cu was released into the environment from

phosphate fertilizer production, agriculture and mining activity, metal and wood

production, and metal waste dumps (Nriagu & Pacynat 1988; ATSDR 2004). High

concentrations of Cu can often be found near waste disposal sites, smelters, mines,

landfills, and industrial settings (ATSDR 2004). Cu does not break down easily in the

- 2 -

environment (ATSDR 2004), thus potentially causing pollution and negative impacts to

the environment.

Acid mine drainage (AMD) is often referred to as acid rock drainage (ARD), caused by

acid drainage from the mine waste rock, tailings and mine structures (pits and

underground workings; U.S. Environmental Protection Agency 1994). The generation of

acid by oxidation of sulphur and the precipitation of ferric iron occurs when the sulphide

and elemental sulphur containing minerals are exposed to the weathering effects of water

and oxygen (Price & Errington 1998). This will result in water acidity, thus causing the

elevated leaching of metals, such as silver, cadmium, arsenic, zinc and copper (U.S.

Environmental Protection Agency 1994), due to high metal solubility and sulphide

weathering rate under acidic conditions (Price & Errington 1998). Metals can travel long

distances when dissolved in water, resulting in the contamination of streams and

groundwater and therefore causing significant environmental impact and threaten the

water sources on which we all depend. AMD has been described as the largest

environmental problem facing the U.S. mining industry (USDA Forest Service 1993;

Ferguson & Erickson 1988; Lapakko 1993). More than 7,000 kilometres of streams

affected by acid drainage from coal mines in the Eastern U.S. have been reported by Kim

et al. (1982). Besides that, according to USDA Forest Service (1993) there are between

20,000 and 50,000 mines currently generating acid, resulting in an acid drainage impact

on 8,000 to 16,000 km of streams (in the Western U.S.). See Table 1.1 for examples of

AMD and some other industrial activities impacting the environment and humans.

- 3 -

Table1.1: Acid mine drainage and some other industrial activities impact towards the environment and human.

Area Heavy Metal Level (ppm) Source of pollution Impact of the pollution Reference

Minamata bay, Japan

(marine products)

Methylmercury

(MeHg) 5.61-35.7

Industrial wastewater

discharged into the

sea

MeHg poisoning in 2252 individuals

over a 36-year period due to the

ingestion of contaminated marine life

Harada (1995)

Ok Tedi, Papua New

Guinea

(river sediment)

Copper

(Cu) 620

Copper and gold

mine tailings

Adverse effect on the livelihood of

50,000 villagers living downstream of

the mine. Damaged ecosystems

covering an area of 2000 km2

Hettler et al.

(1997) Gold

(Au) 0.14-0.28

Nigeria

(Air dust)

Lead

(Pb) 800

Perturbation

by roadside traffic

Adverse effect on the livelihood who

play on the road site and eating raw

fruits and food exposed at dusty

roadsides

Olade (1987)

Bangladesh

(water)

Arsenic

(As) >0.05

Contamination of

drinking water by

naturally occurring

arsenic

35-77 million people from 42 districts

at risk of drinking contaminated

water. In some districts, as high as

57.5% of population had skin lesions

due to arsenic

Smith et al.

(2000)

- 4 -

In Malaysia, there are a few places are known for their Cu pollution such as the Juru River

in Penang (Lim & Kiu 1995) and coastal areas of Peninsular Malaysia (Lukut River;

Ismail & Safahieh 2005). High Cu concentrations (up to 100 µg/g) were recorded from

Lukut River (Ismail & Safahieh 2005) and even higher Cu concentration has been

recorded in Juru River in Penang, with Cu concentrations up to 144 µg g−1 (Lim & Kiu

1995), which is 2 times higher in comparison to the natural average global shale values

(shale value of 45µg g−1) (Mason & Moore 1982). Both studies cited anthropogenic

activities as the source of Cu. See Table 1.2 for more Malaysia local heavy metal

contaminated area.

Table 1.2: Malaysia local heavy metal contaminated area and the source of pollution.

All of the environmental pollution mentioned above may pose risks and hazards to

humans via direct contact or ingestion with the contaminated soil, drinking of the polluted

ground water, and through food chain (polluted water runoff from the mining area into

ocean, bioaccumulation of the heavy metal inside ocean filter feeder such as oyster and

mussle, human consume the polluted oyster and mussle (Okafor & Onwuka 2013). In the

following, we introduce some of the effects that Cu can have on humans.

Area Heavy Metal Level (ppm) Source of

pollution Reference

Ampar Tenang site

Iron 0.97

Waste disposal Rahim et al.

(2010) Lead

(Pb) 0.32

Kelantan River Lead

(Pb) 20.82

Anthropogenic

activity

Ahmad et al.

(2009)

Tanjung Lumpur

Mangrove Forest

(Surface Sediment)

Lead

(Pb) 44.41

Anthropogenic

activity

Yunus et al.

(2011)

Copper

(Cu) 32.79

Anthropogenic

activity

Cobalt

(Co) 5.79 Terigeneous

Manganese

(Mn) 117.73 Terigeneous

- 5 -

1.1.2 Copper Toxicity

Although Cu is one of the essential micronutrients for humans, it can be extremely toxic

if taken in high concentrations. According to Lester (1987), the daily requirement of

copper for an adult is only 0.03 mg/kg and reports of Cu toxicity are becoming more

common these days (Ashish et al. 2013). Symptoms such as vomiting, abdominal pain,

and nausea may occur when one is exposed to acute copper poisoning (Stern et al. 2007;

Ashish et al. 2013). Acute poisoning is rarely seen (Gamakaranage et al. 2011), however,

chronic poisoning can be found happening at those countries, which copper plumbing is

common. For instance, in Germany, all of the patients who suffered from a series of

severe systemic diseases such as copper induced liver cirrhosis and chronic copper

poisoning induced gastrointestinal diseases were found to have copper plumbing that

released copper into the tap water, in their houses. Besides that, the patients were also

recorded to be suffered from nausea, vomiting, colic, and diarrhoea (Eife et al.

1999). Moreover, Ashish et al. (2013) also reported that hepatic necrosis, anaemia,

hypotension, proteinuria, acute renal tubular failure, tachycardia, vascular collapse,

haemoglobinuria, and death may occur when long term exposure to Cu. Table 1.3 below

summarises the impacts of Cu on humans.

Their toxicity depends on:

• Bioavailability: absorption in the gastrointestinal tract, transport to other cell types,

uptake by them.

• Solubility under physiological conditions

• Chemical species: elements are not generally used by cells in their elemental form.

They are used in ionic, complex or organo-metallic forms.

Copper can be found widely distributed in biological tissues and is involved in important

physiological functions in the human body (World Health Organization 1996). It acts as

cofactor for several enzymes in our body such as cytochrome oxidase, ceruloplasmin,

superoxide dismutase, tyrosinase, and dopamine β-hydroxylase (Ashish et al. 2013).

Besides that, it also helps to transmit electrical signals and facilitates the absorption of

iron in our body (Ashish et al. 2013). Insufficient intake of copper may lead to blood

circulation problems, anaemia and growth inhibition (Jennings & Sneed 1996).

- 6 -

Table 1.3: Effects of acute and chronic copper poisoning on human organ.

Human organ Acture Copper

Poisoning

Chronic Copper

poisoning Reference

Systemic Chills, Fever, Pain

Emaciation,

Anaemia, Malaisa,

Debility

Ashish et al. 2013

Eyes and Skin Yellowing

(Jaudice)

Yellowing

(Jaudice) Ashish et al. 2013

Circulatory Anemia, Shock Anemia, Shock Ashish et al. 2013

Gastric

Vomiting, Nausea,

Abdomal pain,

Burning sensation

Vomiting, Nausea,

Diarrhoea, Colic,

Costipation

Ashish et al. 2013

Mouth Metallic taste Metallic taste,

Green line on gums Ashish et al. 2013

Muscular Convulsion, Ashes,

Weakness

Convulsion,

Weakness, Wrist

drop, Foot drop

Ashish et al. 2013

Liver Decreased function Wilson's disease,

Bronze diabetes Ashish et al. 2013

Intestinal Diarrhea Diarrhea Ashish et al. 2013

Kidneys No urine

production Abnormal Ashish et al. 2013

- 7 -

1.2 Current Technologies for Heavy Metal Removal

Unlike organic pollutants, which can be broken down by microbial communities, heavy

metals do not undergo chemical or microbial degradation (Kirpichtchikova et al. 2006).

Besides that, presence of heavy metals can also negatively affect microbial degradation

of organic contaminants (Maslin & Maier 2000). Therefore, other solutions have to be

found to remove heavy metal contaminants.

In recent years, several technologies have been introduced to remove metal contaminants

such as chemical precipitation, phytoremediation (Vaajasaari & Joutti 2006; Wenzel

2009), membrane filtration, ion exchange (Kurniawana et al 2006), electrodialysis

(Mohammadi et al. 2005), photocatalysis (Barakat et al. 2004; Kajitvichyanukula et al.

2005), reverse osmosis (Rich & Cherry 1987), and biosorption (Fourest & Roux 1992).

1.2.1 Chemical Precipitation

Precipitation is one of the common methods that have been used by almost 75% of the

plating companies to extract heavy metals from solutions (Cushnie 1985). The method

works by converting soluble metal salts from the solution into insoluble salts; thereby

precipitating the heavy metal out of the solution. This step is followed by settling

(clarification) and filtration to remove the metal precipitates from the treated solution

effluent (Seneviratne 2007). It was recorded to be simple to operate, cost effective

(Kurniawan et al. 2006), and has proven to effectively remove heavy metals from

electroplating waste water. For instance, Abrego (1997) reported the use of precipitation

and flotation to remove a wide variety of metal ions, such as toxic metal ions nickel (II),

aluminium (III), lead (II), iron (II) and tin (II), within a wide range of pH (3-12) from

contaminated samples. The metal ions were able to be removed below levels required by

anti-pollution regulations (Abrego 1997).

Although the cost for the treatment is known to be low and the operation is simple, it will

result in large volumes of toxic sludge generation, which requires proper disposal;

however, sludge disposal is cost intensive. Besides that, it is difficult to precipitate the

metals held in solution by complexing agents such as EDTA and cyanide. Moreover, the

- 8 -

addition of the reagents has to be carefully controlled in order to avoid unacceptable

concentrations in the treatment effluent (Seneviratne 2007).

1.2.2 Ion Exchange

Ion exchange is one of the common methods that have been used successfully for metal

ion removal from industry effluents. The treatment uses insoluble polymers (resins) that

contain acidic or basic functional groups to exchange the counter-ions from the

surrounding solution. Da̧browski et al. (2004) and Gold et al. (1987) have proven the

simplicity of the method and the efficiency to remove metal ions such as chromium

(III,VI), cadmium (II), mercury (II), vanadium (IV,V), nickel (II), zinc (II), copper(II)

and lead (II), from industrial wastewaters and contaminated electroless. Gold et al. (1987)

also proved that the resins can be high selective. Amberlite IRC-718 resin, for example,

is more selective to copper (94%) than nickel and is relatively selective to zinc and lead

(50%). Duolite ES- 467 resin is on the other hand more selective to zinc and lead (89%)

and relatively selective to copper (75%).

Although ion exchange has been proven to be simple to operate Da̧browski et al. (2004),

efficient in metal removal Gold et al. (1987), low in maintenance cost and requires only

little energy (Griffin 2011), there are several disadvantages that need to be noted. It is pH

sensitive and not able to handle highly concentrated metal solutions (Baysal et al. 2013).

Besides that, it is also easy to be blocked by organics matter and other solids present in

the wastewater such as calcium sulphate and iron (Griffin 2011).

1.2.3 Electrodialysis

Electrodialysis is a process that uses semipermeable ion-selective membranes (Cho et al.

2010) and an electric potential difference to separate the ionic component from a solution

and other uncharged components (Strathmann 1992). This separation process is able to

maintain the metal ions in low concentrations within the anodizing bath solution. The

metal ions are selectively transported through the selective membrane with the electrical

current induced flow (Cho et al. 2010). This method has been used by Pedersen (2003) to

remove cadmium from wood fly ash, with the assisting agent a mixture of ammonium

- 9 -

citrate (0.25 M) and ammonia (1.25%). Besides that, it has also been recorded to be able

to recover silver, copper, nickel, lead, gold, tin, and zinc from cyanide bath rinse solution

(U.S. Environmental Protection Agency 2013).

This electodialysis method is known to have the advantages of high selectivity and

recovery of the metal ions from the solution (Barakat 2011). However, there are several

disadvantages of the method that need to be noted such as the clogging of the membrane

by metal hydroxide formation (Ahalya 2003), the requirement for periodic maintenance,

and technically challenging operation and handling (Barakat 2011).

1.2.4 Semiconductor Photocatalysis

Semiconductor photocatalysis is one of the latest methods used to remove and recover

metal ions from wastewater. It is a process based on reduction by photo generated

electrons and uses ultraviolet (UV) light and semiconductor particles, such as CdS, CeO2,

ZnO, ZnS and TiO2, as catalyst. Electron–hole pairs (e−/h+) are formed in the conduction

and the valence band of the semiconductor, respectively, once the semiconductor particles

are illuminated by UV light with the energy greater than the semiconductor band gap

energy (Herrmann 1999). The charge carriers will then migrate to the surface of the

semiconductor and conduct the reducing/ oxidizing reaction on the component (metal

ions) within the solution. The method has been shown by Barakat et al. (2004) to be able

to remove 78% of free cyanide (10−3 M) within 4 hours of illumination, and free copper

(10−2 M) within an even shorter time (3 h).

The photocatalysis method is known to remove organic and metal pollutants

simultaneously with less harmful by-products. However, it does possess severe

limitations such as a poor overlap of the solar spectrum with the absorption spectrum of

TiO2 (less than 5%). This can be improved by doping the TiO2 with metal ion, which will,

however, significantly increase the cost of the photocatalyst (Malato et al. 2014). Besides

that, there are others limitations such as a limited range of applications and a long duration

time (Barakat 2011). Deactivation of the photocatalyst by strongly adsorbing end

- 10 -

products onto the surface of the photocatalysis can be another limiting factor (Pichat

2013).

1.2.5 Membrane Filtration

Membrane filtration is gaining attention in many industries for its ability to remove

inorganic contaminants, such as heavy metals, from wastewater. Besides that, it is also

known to be capable of removing suspended solid and organic compounds. Various

membrane filtration types can be used for heavy metal removal from wastewater

(depending on the size of the particles), such as ultrafiltration, nanofiltration and reverse

osmosis. (Vigneswaran et al. 2005; Dyson et al. 2008; Wang et al. 2009).

Ultrafiltration (UF) is the use of a permeable membrane to separate suspended solids,

macromolecules, and heavy metals from inorganic solutions. The separation is based on

the basis of the molecular weight (1000-100,000 Da) and pore size (5-20 nm) of the

separating compounds (Vigneswaran et al. 2005). These will allow the small molecule

such as water, which has the size of 0.38 nm (Ngai 2011), to pass through the membrane,

and at the same time, retain the other molecules that have a size larger than the pore size

of the membrane (Sablani et al. 2001).

Nanofiltration (NF), with a membrane pore size of 0.5 – 5 nm (Dyson et al. 2008), uses

separation mechanisms that involve electrical (Donnan) and steric (sieving) effects. A

Donnan potential is created between the charged anion within the membrane and the co-

ions within the effluent, and therefore it creates the conditions to reject the latter ones

(Wang et al. 2009). More specifically, this technique uses the membrane’s small pore size

and surface charges to reject and prevent the charged solutes smaller than the membrane

pore with the bigger neutral ones to pass though the membrane. This technique has been

proven to be able to remove more than 90% of copper ions from industrial feed water

(Qdais & Moussa 2004).

- 11 -

Reverse Osmosis (RO), with a membrane pore size lower than 0.5 nm (Dyson et al. 2008),

is known to be more efficient than UF and NF for heavy metal removal from inorganic

solutions (Wang et al. 2009). The percentage of heavy metal rejection is up to 97% at

metal concentrations between 20-200 mg/L. The technique uses pressure on the heavy

metal solution to force the fluid to pass through the membrane. Therefore, the heavy metal

and purified water will be separated, retained and accumulated on two different sides of

the membrane (Wang et al. 2009). For instance, Benito and Ruíz (2002) were able to

recover up po 95% of the clean water from the polluted water. Qdais and Moussa (2004)

were able to remove 98% of copper and 99% of cadmium from industrial wastewater.

Although the membrane filtration technologies mentioned above shows high recovery of

metal ions and clean water. However, the high operational costs and sensitivity to oxidant,

pH and chlorine, liable fouling, compaction, scaling and limited life of the membrane are

major disadvantages of these technologies (Kurniawan et al. 2006; Wang et al. 2009;

Wang et al. 2010).

1.2.6 Phytoremediation

Phytoremediation is one of the green technologies that uses plants to remove organic and

inorganic pollutants from the environment (Erakhrumen 2007). This technique has been

previously recorded to be used for heavy metal removal from metal contaminated land

(Pulford & Watson 2003) and wastewater (Singh et al. 2012). There are five different

types of phytoremediation that utilise different ways of removing pollutants from the

environment (Wang et al. 2008; Mirsal 2013).

Phytoextraction: A technology that uses hyper-accumulating plants to transport, and to

accumulate contaminants from the soil into the plant roots and aboveground shoots. The

contaminants can be removed by harvesting the plants (Wang et al. 2008; Jadia & Fulekar

2009; Mirsal 2013).

- 12 -

Phytodegradation: A technology that uses plants to degrade or breakdown organic

contaminants mainly through enzymatic reactions (Mirsal 2013).

Rhizofiltration: A technology that uses plant fibrous root systems to absorb, accumulate

and precipitate the contaminants from wastewater (Mirsal 2013; Wang et al. 2008). It is

similar to the phytoextraction technology, however, this technology is mostly used in

aquatic enviroments (Jadia & Fulekar 2009; Mirsal 2013).

Phytostablisation: A technology that uses highly tolerant plants to limit the mobility and

bioavailability of the contaminant in the soil by complexation, precipitation, or sorption

(Wang et al. 2008).

Phytovolatilisation: A technology using plants (mainly trees (Mirsal 2013)) to uptake,

transform and evaporate the contaminant into the atmosphere (Jadia & Fulekar 2009).

Please refer to Table 1.4 for the advantages and disadvantages of each of the five above

mentioned techniques.

Although phytoremediation technology is known to be cost effective and displays high

efficiency in removing metal contaminants, it is pH sensitive, time consuming, has a high

cost and long term maintenance is needed. Besides that, the consumption of the

contaminants polluted plant biomass by herbivores might cause the contaminants to enter

the food chain (Tangahu et al. 2011).

- 13 -

Table 1.4: Overview of advantages and disadvantages of each of the five

phytoremediation techniques /approaches.

Technologies Advantages Disadvantages

Phytoextraction Cost effective, permanent

removal of the contaminant

from soil (Prasad 2004; Wang

et al. 2008)

Time consuming due to slow

growing plants (Prasad 2004;

Wang et al. 2008)

Phytodegradation Able to degrade a wide range

of contaminants (Pivetz 2001)

Possibility of toxic

intermediates or products

formation (Gaspard & Ncibi

2013)

Rhizofiltration Relocation of the system

(plant) is easy (Prasad 2004)

pH sensitive, pre-grown plant

is needed (Prasad 2004)

Phytostabilization Cost effective (Prasad 2004) Long-term maintenance is

required, Contaminants still

remain in the environment

(Prasad 2004; Wang et al.

2008)

Phytovolatilization Converting toxic

contaminants into less-toxic

form, Rapid natural

degradation process (Prasad

2004)

Accumulation of the

contaminant in the vegetation

(Prasad 2004)

1.3 Biosorption

The technologies mentioned above (chemical precipitation, ion exchange, electrodialysis,

photocatalysis, membrane filtration, reverse osmosis, and phytoremediation) are either

expensive, pH sensitive, or easily get fouled Volesky (1990). Besides that, most of those

them may create secondary problems such as toxic sludge formation, and one of them

(phytoremediation) may even cause the contaminant to enter food chains (Tangahu et al.

2011). Therefore, an alternative technology, which involves a biological process between

a solid phase (biological material) and a liquid phase (solvent) containing a dissolved

- 14 -

component to be sorbed (contaminants), has been extensively studied for the past decades

(Volesky 1990). This technology, called biosorption, is known to be highly efficient in

contaminant removal, possesses high contaminant uptake rates, is environmentally

friendly, cost effective, active in a wide range of pH (Volesky 1990; Lee 2014), and

highly selective (Volesky 1990). These advantages have made biosorption so attractive

for the removal of toxic heavy metal contaminants from polluted wastewater. Besides that,

it is also known to be able to regenerate the biosorbent easily (Kratochvil & Volesky 1998)

and the metal can be recovered (Volesky 1990).

Biosorption was defined as the concentration and accumulation of pollutants from

aqueous solutions by the use of biological materials (biosorbent) and therefore allows the

recovery and (or) environmentally acceptable disposal of the pollutants (Dönmez et al.

1999). Biosorption consist of several mechanisms, such as crystallization adsorption,

chelation, precipitation and ion exchange, followed by ion entrapment in intrafi and inter-

brillar capillaries, diffusion through the cell wall and membranes, and spaces of the

polysaccharide material, which vary depending on the origin of the biomass, the

processing steps and the species used (Singh 2006). Biosorption can be divided into two

categories, which are metabolism dependent (active uptake) and metabolism independent

(passive uptake). Metabolism dependent (in which the contaminant will be taken up and

transported across cell membrane) is also known as active biosorption or bioaccumulation

and is associated with cell metabolic activities. On the other hand, metabolism

independent, which is also known as passive biosorption, does not depend on the cell

metabolic activity. It depends on the functions of the chemical composition of the cell

wall. The metabolism-independent biosorption takes place in both live and dead microbial

cells, while metabolism-dependent biosorption can only occur within alive microbial cells

(Lee 2014).

- 15 -

1.3.1 Biosorbents

A wide range of microorganisms such as algae, bacteria, and fungi (live, dead and pre-

treated) have been reported to be able to remove heavy metals. Table 1.5 provides an

overview of previously reported biosorbents.

Table 1.5: Overview of biosorbents and their uptake efficiencies for selected metals.

Biosorbent Species origin Metal Metal Uptake

(mg/g)

Reference

Bacteria

Bacillus

licheniformis Au 59

Beveridge 1986

Streptomyces

nouresei Cr 1.8

Mattuschka et al. 1993

Bacillus subtillis Fe 201 Beveridge 1986

Fungi

Rhizopus

arrhizus Au 164

Kuyucak & Volesky 1988

Trichoderma

viride Cu 1.2

Townsley et al. 1986

Absidia orchidis Pb 351 Holan & Volesky 1995

Penicillium

chrysogenum Cd 56

Holan & Volesky 1995

Algae

Scenedesmus

obliquus Cu 10

Mattuschka et al. 1993

Ascophyllum

nodosum Pb 270 - 360

Holan & Volesky 1994

Sargassum

natans Pb 220 - 270

Holan & Volesky 1994

Fucus

vesiculosus Ni 40

Holan & Volesky 1994

Different forms of biomass (live, dead, pre-treated) will result in different biosorption

rates and capacities. For instance, pre-treated biomass of Aspergillus niger with sodium

carbonate (NA2CO3) has a 5% higher biosorption efficiency than the untreated one, and

31% higher than the biomass treated with hydrochloric acid (Javaid et al. 2011). This is

- 16 -

due to chemical modification of the binding site of the fungal biomass, and thus increased

numbers of active binding sites on the surface area.

Other than microorganisms, plant biomass and mammalian polymers also showed to be

able to biosorb certain metals. For instance, Elifantz and Tel-Or (2002) showed that the

biomass of the Macrophyte, Ludwigia stolonifera, can be used to biosorb- heavy metal

cadmium (Cd) and nickel (Ni). Besides that, Ratnakumari and Sobha (2012) highlighted

the capability of animal polymers, chick and duck feathers, to biosorb the heavy metal

copper (Cu). However, biosorption using microorganisms have been used by most of the

researches (Javaid et al. 2011; Beveridge 1986; Mattuschka et al. 1993; Kuyucak &

Volesky 1988a, Townsley et al. 1986; Holan & Volesky 1995; Holan & Volesky 1994).

Fungal biomass seems to be getting more attention and has been studied more extensively

(Wang et al. 2010). This is mainly due to the reason that fungi possess a wide

morphological variety and can be manipulated morphologically and genetically. Fungal

cell wall is composed of chitosans, glucans, and chitin (Singh 2006). Besides that, it also

contains proteins, lipids, and other polysaccharides. Fungi generate biomass fast due to

short multiplication cycles and can be cultured easily using unsophisticated fermentation

technique (Fulekar 2012; Lee 2014), yielding large quantities of fungal biomass and

derivatives (Lee 2014). Moreover, fungal biomass contains high amounts of cell wall

material such as chitin and chitosan which possess excellent metal-binding properties

(Lee 2014; Gadd 2004). Bishnoi and Garima (2005) and Ahmad et al. (2011) showed that

fungal biomass has better biosorption capacity of heavy metal compared to the other

conventional absorbents such as activated carbon (Nuchar SA) and algae. Fungal biomass

performs better in terms of biosorption of heavy metals compared to ion-exchange resins

which only contain monofunctional groups. This is due to the fact that fungal biomass

contains a much higher variety of functional sites such as sulfate, carboxyl, hydroxyl,

sulfonate, amino, phosphate, imino, sulfydryl, thioether, carbonyl, and imidazole groups

(Singh 2006).

The focus of this thesis is on fungi, in particular endophytic fungi and the following

provides an introduction to the world of endophytes.

- 17 -

1.4 Fungal-plant Symbiotic Interaction

Fossil records indicate that symbiotic interactions between fungi and plants have taken

place since at least 400 million years ago (Krings et al. 2007). A successful fungal-plant

symbiosis involves three different stages:

(a) penetration of the fungus into plant tissues,

(b) colonization of the the host plant tissue by the fungus,

(c) expression of the fungal symbiotic lifestyle (Singh et al. 2011).

There are a few different outcomes of symbiotic interaction as defined by the fitness

benefits realized by both of fungi and the host plant (Lewis 1985). In fungal-plant

symbiosis, the benefits to fungal symbionts can be positive (parasitism, commensalism

and mutualism), neutral (neutralism and amensalism) or negative (competition), while for

the host plant it can also be positive (mutualism), neutral (neutralism and commensalism)

or negative (parasitism, amensalism and competition; Rodriguez et al. 2008).

1.4.1 Endophytic Fungi

An endophytic fungi is a fungus that lives symbiotically with a host plant without showing

any apparent symptoms. Endophytic fungi protect their host plant from biotic and abiotic

stress such as increasing their stress tolerance (for example against drought, salinity, and

heavy metals). They can enhance the growth of their host plants by reducing the infection

rate of nematodes and defending the plant from diseases (Tadych & White 2009; Sikora

et al. 2008; Varma et al. 1999; Redman et al. 2002). In return, they will acquire nutrients

from their host plant (Tadych & White 2009). There are two different mechanisms

involved in the endophytic fungi-conferred stress tolerance, which are (a) rapid activation

of host stress response systems after stress exposure of the symbiotic host plants (Redman

et al. 1999) and (b) synthesis of anti-stress biochemicals in the host plant, either through

endophytic fungi induction or by the endophytic fungi itself (Bacon & Hill 1996).

However, the details of how the endophytic fungi activate their host stress tolerance

/response still remains a mystery (Rodriguez et al. 2004). Many studies conducted all

around the world, have proven that endophytic fungi significantly contribute to or are

responsible for the adaptation of their host plant towards environmental stresses such as

drought, extreme temperature, high salinity, heavy metal toxicity, and oxidative stress

- 18 -

(Malinowski et al. 1997; Redman et al. 2001; Rodriguez et al. 2004; Rodriguez et al. 2008;

Soleimani et al. 2010; Monnet et al. 2001; Ren et al. 2011; Rodriguez & Redman 2008).

The endophytic fungus Penicillium minioluteum LHL09, isolated from soyabean plant

(glycine max. L.) was shown to be able to protect the host plant from abiotic salinity stress

(Khan et al. 2011). Besides that, Ren et al. (2011) and Soleimani et al. (2010) also

demonstrated the capability of endophytic fungi to increase the resistance of their host

plants against heavy metal cadmium. Moreover, Monnet et al. (2001) have shown the

capability of the endophytic fungus Neotyphodium lolii to increase zinc tolerance in

Lolium perenne. Table 1.6 summarise previously reported endophytic fungi-plant

interaction.

Table 1.6: Overview of endophytic fungi and their host plant interaction.

Protection

from Endophytic fungi Host plant Reference

Drought

stress

Neotyphodium sp. Festuca pratensis Malinowski et al.

1997

Acremonium

coenophialum

Festuca

arundinacea

Schreb.

Elbersen & West

1996

Curvularia

protuberate

(Cp4666D)

Dichanthelium

lanuginosum Rodriguez et al. 2008

Colletotrichum magna

(L2.5)

Lycopersicon

esculentum Redman et al. 2001

Colletotrichum

orbiculare (683)

Lycopersicon

esculentum Redman et al. 2001

Colletotrichum

gloeosporioides (95-

41A)

Lycopersicon

esculentum Redman et al. 2001

Colletotrichum magna

(path-1) Capsicum annuum Redman et al. 2001

- 19 -

Colletotrichum sp. Lycopersicon

esculentum Rodriguez et al. 2004

Piriformospora indica Arabidopsis sp. Sherameti et al. 2008

Fusarium sp. Lycopersicon

esculentum

Rodriguez & Redman

2008

Alternaria sp. Lycopersicon

esculentum

Rodriguez & Redman

2008

Salinity stress Fusarium culmorum

(FcRed1) Leymus mollis Rodriguez et al. 2008

Parasitic

nematodes Fusarium oxysporum

Banana, tomato

and rice Sikora et al. 2008

Parasitic

nematodes Trichoderma

Banana, tomato

and rice Sikora et al. 2008

Heavy metal

stress

Endophytic fungi Fine fescues Zaurov et al. 2001

Neotyphodium

Festuca

arundinacea and F.

pratensis

Soleimani et al. 2010

Endophytic fungi Lolium

arundinaceum Ren et al. 2011

Neotyphodium lolii Lolium perenne Monnet et al. 2001

Heat stress

Fusarium sp. Lycopersicon

esculentum

Rodriguez & Redman

2008

Alternaria sp. Lycopersicon

esculentum

Rodriguez & Redman

2008

Curvularia

protuberata

Lycopersicon

esculentum Rodriguez et al. 2008

Curvularia sp. Lycopersicon

esculentum

Rodriguez & Redman

2008

Curvularia

protuberate

Dichanthelium

lanuginosum Redman et al. 2002

- 20 -

1.4.2 Heavy Metal Tolerance of Endophytic Fungi

Many studies have been proven that fungi are able to tolerate a wide range of heavy metals

(Carrillo-González & González-Chávez 2012; Hegedűs et al. 2007; Weissenhorn et al.

1993; Gaur and Adholeya 2004; Zafar et al. 2007), including endophytic fungi (Ban et al.

2012; El-Gendy et al. 2011; Khan & Lee 2013). Recent studies have shown the capability

of the endophytic fungi to be able to tolerate several heavy metals. For instance,

Penicillium funiculosum LHL06 and Metarhizium anisopliae, isolated from the roots-

tissues of soybean plants, were recorded to be able to tolerate copper (Khan & Lee 2013).

Besides that, Ban et al. (2012) also demonstrated the capability of the fungal endophytes

Cladosporium cladosporioides, Gaeumannomyces cylindrosporus, and Exophiala

salmonis, to tolerate copper, lead, and zinc. See Table 1.7 for more previously reported

examples.

Table 1.7: Overview of endophytic fungi and their resistance against heavy metal.

Endophytic fungi Heavy metal tolerance Reference

Penicillium funiculosum LHL06 Copper Khan & Lee 2013

Metarhizium anisopliae Copper Khan & Lee 2013

Gaeumannomyces

cylindrosporus Lead, Copper and Zinc

Ban et al. 2012

Paraphoma chrysanthemicola Lead, Copper and Zinc Ban et al. 2012

Phialophora mustea Lead, Copper and Zinc Ban et al. 2012

Cladosporium cladosporioides Lead, Copper and Zinc Ban et al. 2012

Exophiala salmonis Lead, Copper and Zinc Ban et al. 2012

Rhizopus oryzea Copper and Cadmium El-Gendy et al. 2011

Aspergillus luchuensis Copper and Cadmium El-Gendy et al. 2011

Monacrosporium elegans Copper and Cadmium El-Gendy et al. 2011

Curvularia lunata Copper and Cadmium El-Gendy et al. 2011

Penicillium lilacinum Copper and Cadmium El-Gendy et al. 2011

Drechslera hawaiiensis Copper and Cadmium El-Gendy et al. 2011

Verticillium Fungicola Copper and Cadmium El-Gendy et al. 2011

Pestalotiopsis clavispora Copper and Cadmium El-Gendy et al. 2011

- 21 -

1.4.3 Biosorption of Heavy Metal using Endophytic Fungi

Besides the capability to tolerate heavy metals, endophytic fungi have also been recorded

to be powerful biosorbents of heavy metal using either live or dead biomass. For instance,

Microsphaeropsis sp. LSE10, isolated from Solanum nigrum L., showed the capability to

biosorb cadmium (Xiao et al. 2010). Two other endophytic fungi, Metarhizium anisopliae

and Penicillium funiculosum LHL06 (collected from the roots-tissues of soybean plants)

were recorded to be able to biosorb copper and cadmium (Khan & Lee 2013). Mucor sp.

CBRF59, which is an endophytic fungus isolated from Brassica chinensis, was able to

biosorb cadmium and lead by using its live and dead biomass (Deng et al. 2011). See

Table 1.8 for more previously reported examples.

Table 1.8: Overview of endophytic fungi and their capability of biosorpt heavy metal.

Endophytic fungi Heavy metal (Biosorption) References

Metarhizium anisopliae Copper and Cadmium Khan & Lee 2013

Penicillium

funiculosum LHL06 Copper and Cadmium Khan & Lee 2013

Lasiodiplodia sp. MXSF31 Lead, Zinc and Cadmium Deng et al. 2014

Mucor sp. CBRF59 Cadmium and Lead Deng et al. 2014

Microsphaeropsis sp. LSE10 Cadmium Xiao et al. 2010

Rhizopus oryzea Copper and Cadmium El-Gendy et al. 2011

Aspergillus luchuensis Copper and Cadmium El-Gendy et al. 2011

Monacrosporium elegans Copper and Cadmium El-Gendy et al. 2011

Curvularia lunata Copper and Cadmium El-Gendy et al. 2011

Penicillium lilacinum Copper and Cadmium El-Gendy et al. 2011

Drechslera hawaiiensis Copper and Cadmium El-Gendy et al. 2011

Verticillium Fungicola Copper and Cadmium El-Gendy et al. 2011

Pestalotiopsis clavispora Copper and Cadmium El-Gendy et al. 2011

- 22 -

1.5 Pitcher plants (Nepenthes) as Source of Endophytic Fungi

Nepenthes, also known as pitcher plant or monkey cup, is a type of carnivorous plant

under the family of Nepenthaceae (Adam 1997). Hundreds of species can be found

(McPherson 2009) and several studies have addressed the enzymatic properties of the

digestive fluid, trapping mechanism, and geological distribution (Adam et al. 1992;

Merbach et al. 2001; Mithöfer 2011; Slack & Gate 2000; Adam 1997; Lambers & Colmer

2006). There are, however, only very limited studies regarding endophytic fungi in

Nepenthes and one of the main aims of this thesis is to provide some baseline information

on their occurrence. Chapter 3 provides a more detailed introduction to the topic and in

the following, a short introduction to Nepenthes is provided.

Nepenthes are known to have a remarkable modified leaf that consists of a tube-shaped

cup (pitcher) at the end of the tendril. The pitcher can be divided into three different zones,

which are:

(a) Peristome, which is known to be involving in attracting and trapping prey (Figure 1.1

(Clarke 1997); Mithöfer 2011).

(b) Slippery and waxy inner zone, which used for to trap and prevent the prey from

escaping (Figure 1.1 (Clarke 1997); Gaume et al. 2002).

(c) Digestive zone, which is fully covered with glands and filled with digestive fluid

(Figure 1.1 (Clarke 1997); Gorb et al. 2004; Mithöfer 2011).

Figure 1.1: Basic structure of Nepenthes pitcher.

- 23 -

1.5.1 Distribution

Arthropods, especially insects, are attracted by the extrafloral nectar produced by

nectaries located at peristome of the pitcher (Figure 1.1; Merbach et al. 2001). A study

carried out by Kurup and the team (2013) showed that the peristome of Nepenthes

khasiana emits a distinct blue fluorescence under UV (366 nm), and it is believed that the

fluorescence is used to attract the prey. Reduction of the prey capture in the Nepenthes

khasina pitchers was observed when the blue emissions of the peristome were masked.

Once the prey in trapped and drowned within the pitcher, the digestive glands at the inner

wall of the pitcher will secrete digestive fluid to breakdown the prey. A wide variation of

the enzymes were found within the digestive fluid, such as lipase, ribonuclease, acid

phosphatase protease, and esterase (Slack & Gate 2000).

Nepenthes are widely distributed in Borneo, Peninsular Malaysia, and Sumatra, such as

Nepenthes gracilis, Nepenthes mirabilis, and Nepenthes ampullaria and several species

(Nepenthes rajah, Nepenthes xalisaputrana, Nepenthes villosa; Adam et al. 1992; see

Figure 1.2).

- 24 -

Figure 1.2: Distribution map of Nepenthes sp., taken from Carnivorous Plants /

Insectivorous Plants in the Wilderness.

1.5.2 Habitat

Nepenthes can be found growing from sea level to 3,400 m above sea level and have been

recorded living in soils that are low in macronutrients and some were even found growing

on soil containing toxic heavy metals (Adam 1997). For example, Nepenthes northiana,

clipeata and mapuluensis were found growing on nutrient-poor soil of the limestone

habitat (Adam 1997). Nepenthes rajah, xalisaputrana and villosa were found growing in

serpentine habitats where the soils are very low in available macronutrients and rich in

toxic heavy metal such as chromium, magnesium and nickel (Lambers & Colmer 2006).

However, our understanding of the strategy used by the plants to survive in heavy metal

contaminated soils is still very limited. One possible reason could be the association with

metal-tolerant endophytic fungi and a recent study by Lee et al. (2014) showed that N.

mirabilis and N. ampullaria were host to a wide range of endophytic fungi such as

Aspergillus terreus, Sarcosomataceae, Trichoderma asperellum, Isaria, Colletotrichum

gloeosporioides, Penicillium simplicissimum, and Lasiodiplodia.

- 25 -

Chapter 4 studied the ability of Nepenthes-associated endophytic fungi.

1.6 Proteomics - Regulation of Fungi Proteins in Response to Heavy Metal Stress

The proteome, defined as a complete set of the proteins expressed by a genome (Wilkins

et al. 1996) and “the proteins present in one sample (tissue, organism, and cell culture) at

a certain point in time” (Ravi et al. 2013), is dynamic and different from the genome,

which is relatively static. Every organism has only one unique genome, the proteome,

however, can be varied and even result in different phenotypes. For example, three

different stages of beetle life cycle (larve, pupa, and beetle) share one common genome

but vary in proteomes. The proteome often undergoes changes in response to the extra-

and intracellular environmental signals (Rastogi et al. 2006; Ravi et al. 2013), and all of

the changes can be studied through proteomics, the study of the proteome (Pandey &

Mann 2000).

Heavy metal induced oxidative stress will cause cellular damages to proteins (Letelier et

al. 2005), lipids (Zhao et al. 2014), and nucleic acids (Linder 2012) within an organism.

In order to protect itself from the damages and survive through the stress conditions, fungi

are known to regulate certain types of proteins within the cell which are involved in cell

protection (Yιldιrιm et al. 2011). This regulation of the proteins can be studied and

understood by using the proteomics approach (Jensen 2006). Some of the common

proteins and enzymes involved in cell protection are:

(a) Antioxidant enzyme - helps to protect against oxidative cellular damage (Angelova et

al. 2005).

(b) Heat shock protein - serves as molecular chaperones that play an important role in

protein-protein interactions and prevent against protein mis-folding and aggregation

(Borges and Ramos 2005; Csermely & Yahara 2003).

(c) DNA repairing enzyme - repairs errors occurring during DNA recombination and

replication (Mol et al. 1995).

- 26 -

The proteins mentioned above (a, b, c) are examples of proteins known to be upregulated

when exposed to heavy metal stress. For example, a white rot fungus, Phanerochaete

chrysosporium, is known to produce heat shock protein and DNA repairing enzyme in

response to lead exposure. Besides that, (Azevedo et al. 2007) also demonstrated the

activation of the antioxidant defence system in two aquatic fungi, Heliscus submersus and

Varicosporium elodeae, in response to zinc and copper stress.

Proteomics allows quantitative and quanlitative measurements of the fungal proteins and

the information obtained is important for our understanding of proteins involved in

cellular processes. This method allows an accurate analysis of cellular system changes in

response to different copper concentration.

1.7 Aims of the Present Study and Dissertation Outline

The first aim of the present study is to assess the Nepenthes ampullaria associated

endophytic fungi tolerance against the heavy metal copper and to evaluate for their

biosorption capacity.

The second aim is to compare the two groups of endophytic fungi isolated from Nepenthes

ampullaria plants collected from undisturbed and anthropogenically affected areas

(Mentawai Jungle and Kota Samarahan roadside, Kuching) on their heavy metal

resistance and biosorption capacity of removing heavy metal (Cu) from solution using

their Live and Dead biomass.

The third aim is to study and to understand the differentially expressed proteins of the

best fungal isolate (NA40; achieved the highest biosorption capacity using its live

biomass) in response to treatments with 3 different concentration of copper (0, 300, and

500 ppm).

- 27 -

The objectives of this study are:

Chapter 3

i. Isolation of endophytic fungi from Nepenthes ampullaria collected from

undisturbed and anthropogenically affected areas; Mentawai Jungle and Kota

Samarahan roadside, Kuching.

ii. Preliminary screening of the isolated fungal against their heavy metal copper

tolerance.

iii. Identification of the top 11 fungal isolates that were able to survive up to 1,000

ppm of heavy metal copper

iv. Evaluation of the chosen fungi (11) for their biosorption capacity on heavy metal

copper by using the Live and Dead biomass

Chapter 4

v. Proteomics analysis of the best fungal isolate (NA40; achieved the highest

biosorption capacity using its live biomass) on its differentially expressed proteins

in response to heavy metal copper.

- 28 -

Chapter 2

Methodology

2.1 Sampling Sites

Nepenthes ampullaria plant samples were collected at two different sites (see Table 2.1

for Global Positioning System (GPS) coordinates):

1. Ulu Mentawai (located at northern part of Gunung Mulu National Park)

2. Kota Samarahan road site, Kuching, Sarawak, Malaysia.

Table 2.1: GPS coordination for the Nepenthes ampullaria plant samples collected.

Nepenthes ampullaria Global Positioning System (GPS)

Mentawai Jungle N04o 14' 39.3'' E 114o 52' 04.0''

Mentawai Jungle N04 o 14' 39.4'' E 114o 52' 03.9''

Mentawai Jungle N04o 14' 39.0'' E 114o 52' 04.3''

Mentawai Jungle N04o 14' 39.1'' E 114o 52' 04.4''

Kuching Kota Samarahan Roadside 1.501992, 110.392635

Figure 2.1 and 2.2 show the location maps were the Nepenthes ampullaria plants were collected.

- 29 -

Figure 2.1: Ulu Mentawai (sampling sites), located at northern part of Gunung Mulu National Park, indicated by red point (Source: Google Map).

Figure 2.2: Kuching Kota Samarahan roadside (sampling sites), indicated by red point (Source: Google Map).

- 30 -

2.1.1 Ulu Mentawai

In the year of 2012, the Heart of Borneo Initiative, which is a NGO-supported and

government-led programme, organised a scientific expedition to Ulu Mentawai, located

at northern part of Gunung Mulu National Park (Figure 2.1). The area is well-known as

world heritage site that consist mainly of karangas forest and lowland mixed dipterocarp

forest.

The Nepenthes ampullaria plant samples (Figure 2.3) were collected (Figure 2.4) during

the expedition.

Figure 2.3: Nepenthes ampullaria, photographed on site (Mentawai jungle).

- 31 -

Figure 2.4: Author collecting plant samples collecting at in Mentawai jungle.

The Mentawai expedition was held from 5-17 September 2012, in total there are 5 plants

collected from the jungle: 3 Nepenthes ampullaria and 2 Nepenthes rafflesiana

(identification with the kind help of staff from Sarawak Forestry Department). However,

only the endophytic fungi isolated from the Nepenthes ampullaria were used in the

experiments.

2.1.2 Kota Samarahan Roadside

Another Nepenthes ampullaria collection was carried out at the main roadside of Kota

Samarahan (Figure 2.5), located at south east of Kuching. The plants were growing

besides the main road and very closely to the peat swamp. The plant sampling at Kota

Samarahan roadside was done on 2nd January 2013. Only 1 Nepenthes ampullaria plant

(identification done base on the plant and pitcher morphology; Lloyd 1942) was collected

for endophytic fungi isolation.

- 32 -

Figure 2.5: Kuching Kota Samarahan Roadside, the area where the Nepenthes ampullaria

was collected.

2.2 Nepenthes ampullaria Associated Endophytic Fungi Isolation

2.2.1 Ulu Mentawai

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

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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)

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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

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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

Kota Samarahan, Sarawak, Malaysia.

*Corresponding author

Email: cgwong@swinburne.edu.my

Phone number: +60168716911

ABSTRACT

Biosorption using biological materials 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 (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) evaluated for their biosorption capacity

(live and dead biomass). In total, 147 fungal isolates were isolated from Nepenthes

ampullaria and only 7.5% (11) of the total isolates were capable to resist copper

concentration up to 1000 ppm. The 11 fungal isolates were identified through molecular

method, and all of them were grouped with members of the Phomopsis, Diaporthe,

Nigrospora, and Xylaria. The fungal isolate NA40 that was related to Xylaria sp. achieved

the highest biosorption capacity of of 73.26 mg/g using live biomass, while the isolates

NA41 that was related to Phomopsis sp. had the highest biosorption capacity of 59.33

mg/g using dead biomass.

Keywords: Endophytic fungi, Biosorption, Heavy metal copper, Nepenthes ampullaria

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3.1 Introduction

Nepenthes, also known as pitcher plant or monkey cup, is a carnivorous plant that consists

of a tube shaped cup at the end of the tendril filled with digestive fluid to trap and digest

other living organisms and to absorb their nutrients (Hua & Li 2005). Nepenthes (N.) are

widely distributed in Borneo, Peninsular Malaysia and Sumatra, such as N. gracilis, N.

mirabilis, and N. ampullaria and several species (N. rajah, N. xalisaputrana, N. villosa)

have been recorded living in soils low in macronutrients and containing toxic heavy

metals such as chromium (Cr), magnesium (Mg) and nickel (Ni) (Adlassnig et al. 2005).

However, our understanding of the strategy used by the plants to survive in heavy metal

contaminated soils is still very limited. Soleimani et al. (2010) studied the effect of

Neotyphodium, an endophytic fungus, on cadmium (Cd) tolerance of two grass species

(Festuca arundinacea and Festuca pratensis) and showed that endophyte-infected plants

had a higher Cd tolerance compared to non-infected plants. Ren et al. (2011) also

demonstrated that endophytic fungus-infected tall fescue (Lolium arundinaceum) had a

higher Cd tolerance compared to the non-infected one. It seems likely that the presence

of the endophytic fungi in the plants – the symbiosis between endophyte and plant – could

be the reason behind the observed tolerance of the carnivorous plants towards the heavy

metal.

Copper is one of the essential micronutrients for human as it helps to transmit electrical

signals and facilitates the absorption of iron in our body (Ashish et al. 2013). However,

it can be extremely toxic in high concentration. Symptoms such as abdomen pain, purging

and vomiting will be observed when taken in excess (Ashish et al. 2013). Copper (Cu)

can be found naturally occurring in the environment. It was also found to be released into

the environment by human activities such as phosphate fertilizer production, metal and

wood production, agriculture and mining activity. Copper does not break down easily in

the environment, thus causing the environmental pollution such as acid mine drainage

(AMD). The leaching of metal (Cu is one of the metals present) by AMD at mining area

into the water and sentiment heavily affects the environment, rendering the heavily

polluted areas unsustainable to life (ATSDR 2004; McCarthy 2011; Johnson & Hallberg

2005). In Malaysia, few copper pollutions have been reported such as the copper pollution

of the Juru River (located in Penang) that was caused by pig farm discharges and wastes,

with Cu concentrations up to 144 µg g−1 (Lim & Kiu 1995), which is 2 times higher in

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comparison to the natural average global shale values (shale value of 45µg g−1) (Mason

& Moore, 1982). Besides that, high Cu concentrations (37 - 100 µg/g) have also been

recorded in coastal areas of Peninsular Malaysia (Lukut River), with up to 60% of the

copper the results of previous anthropogenic activities (Ismail & Safahieh 2005).

In recent years, several procedures have been designed to deal with heavy metal

contamination of rivers and streams, for example chemical precipitation,

phytoremediation (Vaajasaari & Joutti 2006; Wenzel 2009), ion exchange, reverse

osmosis, solvent extraction (Iyer 1990) and biosorption (Fourest & Roux, 1992).

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

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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

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(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.

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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

specialized taxonomical expertise (Gherbawy & Voigt 2010).

All the selected isolates were successfully identified using molecular method through

sequence homology of their Internal Transcribed Spacer (ITS) genes. The majority of the

isolates were grouped with members of the Phomopsis, followed by Diaporthe,

Nigrospora, and Xylaria (see Table 3.2 for an overview of closest matches and Figure 3.1

for the phylogenetic tree). From the fungal isolates identification, all of the identified

fungi genus (Phomopsis, Diaporthe, Nigrospora, and Xylaria) have been previously

reported as endophytic fungi.

Most of the sequences (55% - NA25, NA27, NA28, MNA3 and MNA27) obtained were

closely related to the fungal genus of Phomopsis. Phomosis sp. were previously found to

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be plant pathogens that can cause foliar infections (Sharma & Florence 1997) and stem

cankers (Hadi & Nuhamara 1997) to some plants. However, our results show that

Pomopsis sp. appeared as Nepenthes ampullaria associated endophytic fungi. Similar

results were also obtained from an endophytic fungi study carried out by Lee and his team

(2014) which revealed that Phomopsis were isolated from Nepenthes plants. Besides that,

Phomopsis sp. also appeared as endophytes of the Tripterygium wilfordii plant (Kumar

& Hyde 2004), Spondias mombin plant (Rodrigues et al. 2000) and Garcinia plant

(Phongpaichit et al. 2007). Phomopsis have been previously recorded to be able to tolerate

heavy metals such as aluminium, lead, and chromium, with a copper tolerance up to 500

ppm (Sim et al. 2015). Our study highlights the higher tolerance of the Phomopsis sp.

against the heavy metal up to 1000 ppm, which is 2 times higher compared to the previous

study.

Three (27% - NA31, S1, S2) of the fungal isolates were grouped under Diaporthe sp..

Diaporthe is a teleomorph form of Phomopsis (Santos & Phillips 2009). Diaporthe

phaseolorum was previously recorded to be a fungal parasite in plants and also involved

in cutaneous infections in humans (Mattei et al. 2013). Some studies have shown that

Diaporthe phaseolorum can also appear as an endophyte in certain plants such as the

medicinal plant Baccharis trimera (Vieira et al. 2014), Laguncularia racemosa

(Sebastianes et al. 2012) and Taxus wallichiana var. mairei (Zaiyou et al. 2013).

Diaporthe are poorly understood in terms of their heavy metal tolerance, however,

Tomono and team (1982) have demonstrated that pycnospores of Diaporthe citri are able

to germinate with a germination rate of 22% under 10-5M (16 ppm) copper solution. Our

study shows the capability of Diaporthe to tolerate copper up to 1000 ppm.

Only 18% of the fungal isolates were grouped under the genus of Nigrospora (9%) and

Xylaria (9%). Both of the Nigrospora sphaerica and Xylaria sp. have been widely

reported as endophytes. For example, Senthilkumar and team (2014) reported endophytic

Nigrospora sphaerica isolation from tropical tree species (Tectona grandis L.) of India,

and Gallo and the team (2009) also reported isolation of endophytic Nigrospora sphaerica

from an Andean plant (Smallanthus sonchifolius). Xylaria species have a very wide

geographical distribution (Patil et al. 2012, Stadler et al. 2014). They have also been found

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as endophyte in plants such as orchids (Chen et al. 2013), ausubo and Australian pine

(Bayman et al. 1998). Besides that, they have also been recorded as wood decay fungi

and found to be associated with termite nests (Ju & Hsieh 2007). In this study, the isolated

fungi NA8 and NA40 had the closest match (99% match over 510/514 bases) with

Nigrospora sphaerica strain BTN39 [KM510416] and with Xylaria sp. 4Y-Cs2-

1[AB741621; 87% match over 279/319 bases], respectively.

Table 3.2: ITS phylogenetic results for the 11 isolated fungi strains.

Sequence

(Fungal Isolate) Closest match Identities

Phylogenetic

division

NA8 Nigrospora sphaerica strain

BTN39 [KM510416]

510/514

(99%) Nigrospora

NA25 Phomopsis sp. 45GP/T

[GQ352480]

525/527

(99%) Phomopsis

NA27 Phomopsis sp. 45GP/T

[GQ352480]

536/538

(99%) Phomopsis

NA28 Phomopsis sp. 45GP/T

[GQ352480]

528/530

(99%) Phomopsis

NA31 Diaporthe sp. P051

[EF423532]

528/530

(99%) Diaporthe

NA40 Xylaria sp. 4Y-Cs2-1

[AB741621]

279/319

(87%) Xylaria

NA41 Uncultured fungus

[FR863605]

531/532

(99%) Phomopsis

S1 Diaporthe sp. 99AS/S

[GU066666]

538/538

(100%) Diaporthe

S2 Diaporthe sp. 99AS/S

[GU066666]

538/538

(100%) Diaporthe

MNA3 Phomopsis sp. M31

[HM595507]

535/536

(99%) Phomopsis

MNA27 Fungal sp. mh500.1

[GQ996123]

540/540

(100%) Phomopsis

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Figure 3.1: ITS gene-based phylogenetic tree representing fungal sequences conserved

within the internal transcribed spacer region. The phylogenetic tree was constructed using

Mega 6 with distance method and sequence distances were calculated using maximum

likelihood method. Boot strap values of 2000 are shown and the cut off value of 50%.

Diaporthe sp. 99AS/S (GU066666)

S1

Diaporthe sp. 60AS/S (GU066638)

Diaporthe phaseolorum isolate 58AS/S (GU066637)

S2

NA31

Diaporthe sp. P051 (EF423532)

Fungal endophyte culture-collection STRI:ICBG-Panama:TK1637 (KF435291)

Uncultured fungus (FR863605)

NA41

Phomopsis sp. 179GP/T (GQ352484)

Phomopsis sp. HNY29-2B (KF387574)

Phomopsis longicolla (EU236702)

Fungal sp. mh500.1 (GQ996123)

MNA27

Phomopsis sp. C1c7b (JX436795)

Diaporthe hongkongensis strain HNCQ1 (KJ609019)

Phomopsis sp. M31 (HM595507)

Diaporthe sp. 138SD/T (GU066697)

MNA3

Phomopsis sp. RP78 (JF441186)

Phomopsis sp. 122AC/L (GU066685)

Phomopsis sp. 45GP/T (GQ352480)

NA25

NA27

NA28

Phomopsis sp. OU-E 208 (KM668705)

NA8

Nigrospora oryzae strain F9 (KM979813)

Nigrospora oryzae isolate TR171 (HQ608152)

Nigrospora sphaerica strain BTN39 (KM510416)

NA40

Xylaria cubensis (AB625431)

Xylariaceae sp. 4Y-Cs2-1 (AB741621)

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Biosorption of Copper by Fungal Live Biomass

As can be seen in Table 3.3, all the live biomass of the isolated fungi (Phomopsis,

Diaporthe, Nigrospora, and Xylaria) have been shown to biosorb metal ion. Fungal

isolate NA40 had the highest biosorption capacity of 73.26 mg/g and it was molecularly

identified to be closely related to Xylaria sp. 4Y-Cs2-1 [AB741621]. Xylaria sp. have

been previously studied for their capability of degrading lignin (Pointing et al. 2003) and

the pollutant polycyclicaromatic hydrocarbons (benzo(a)pyrene) (Chang et al. 2007). Our

research newly uncovered the ability of Xylaria sp. to tolerate the heavy metal copper up

to 1000 ppm, and to remove the heavy metal from the liquid solution by using live and

dead biomass. To the best of our knowledge, this is the 1st report of Xylaria sp. in regards

to copper tolerance and biosorption of copper.

The isolated fungus NA41 achieved the 2nd highest biosorption capacity of 71.34 mg/g,

followed by the other fungal isolates NA27, NA25, NA28 and MNA27, which had live

biomass copper biosorption capacities of 71.34, 59.81, 52.70, 52.31, 36.47 mg/g,

respectively (Table 3.3). Fungi MNA3 had the lowest biosorption capacity of 13.42mg/g.

All of the 6 fungal isolates mentioned above were grouped with Phomopsis. Phomopsis

have been previously reported to be able to degrade toxic recalcitrant N-heterocyclic

compounds (Indole and its derivatives) and remove heavy metal zinc from liquid solution

by its active biomass with a bioadsorption capacity of 0.055 mg/g ± 0.005 of dry weight

(Wang et al. 2014; Alozo et al. 2002). In our research, the isolates related to Phomopsis

sp. showed to be capable of removing heavy metal copper from the liquid solution at a

much higher rate.

Nigrospora sp. have been previously reported to have the ability of degrading hop bitter

acids (Huszcza et al. 2008), and toxic synozol red dye into non-toxic metabolites that are

safe for plant growth (Ilyas et al. 2013). A marine-derived Diaporthe sp. was found to be

able to decolorize and detoxify the raw textile effluent that contains toxic dyes such as

azo dye (Verma et al. 2010). As mentioned above, Phomopsis, an anamorph form of

Diaporthe, was found to be able to remove heavy metal zinc from liquid broth. Besides

that, the heavy metal copper ion absorption into the cytoplasm within the pychospore of

Diaporthe citri has been reported by Tomono et al. 1982, however, no specific amount of

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adsorpted copper ion was reported. Fungal isolated fungi that were related to Diaporthe

were able of removing heavy metal copper from liquid solution by the live biomass with

copper biosorption capacities of 34.17, 43.89, 44.53 and 15.75 mg/g live biomass,

respectively (Table 3.3).

Table 3.3: Biosorption capacity (mg/g) of the Live biomass of the isolated fungal on heavy metal copper biosorption.

Fungal

Isolates

Biosorption of Heavy metal

copper using Live Biomass

(mg/g)

Closest match

NA8 15.75 Nigrospora sphaerica strain BTN39

[KM510416]

NA25 52.70 Phomopsis sp. 45GP/T

[GQ352480]

NA27 59.81 Phomopsis sp. 45GP/T

[GQ352480]

NA28 52.31 Phomopsis sp. 45GP/T

[GQ352480]

NA31 34.17 Diaporthe sp. P051

[EF423532]

NA40 73.26 Xylaria sp. 4Y-Cs2-1

[AB741621]

NA41 71.34 Uncultured fungus

[FR863605]

S1 43.89 Diaporthe sp. 99AS/S

[GU066666]

S2 44.53 Diaporthe sp. 99AS/S

[GU066666]

MNA3 13.42 Phomopsis sp. M31

[HM595507]

MNA27 36.47 Fungal sp. mh500.1

[GQ996123]

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Biosorption of Copper by Fungal Dead Biomass

The capability of the biosorption of heavy metal copper by using the dead biomass of the

isolated fungi (Phomopsis, Diaporthe, Nigrospora, and Xylaria) have been studied and

all of the isolates showed to be able to biosorb the heavy metal copper by their dead

biomass (Table 3.4). The fungal NA41 whichhad the closest match with an uncultured

fungus [FR863605] – phylogenetic division of Phomopsis (99% match over 531/532

bases), showed to have the highest biosorption capacity of 59.33 mg/g. The fungal isolate

NA25 that had the closest match with Phomopsis sp. 45GP/T [GQ352480; 99% match

over 525/527 bases], however, had the lowest biosorption capacity of copper (9.87 mg/g).

Apart from that, the other fungal isolates (NA27, NA28, MNA3, MNA27) that were all

grouped with the same genus, Phomopsis, showed to have copper biosorption capacities

of 11.05, 15.42, 23.07, and 39.73 mg/g, respectively (Table 3.4). Phomopsis sp. have been

previously shown to have the capability to bioadsorb 179 mg/g of lead, 26 mg/g of

cadmium, 6 mg/g of nickel, 10 mg/g of zinc and 25 mg/g of copper into the insoluble

polysaccharidic biomaterial, with the chitosan and glucans as the main components

(Saiano et al., 2005). Besides that, Sim et al. (2015) also demonstrated that the biosorption

of the heavy metal using the dead biomass of Phomopsis sp. had a biosorption capacity

of 19.6 of lead, 20.1 mg/g of cadmium, 18.1 mg/g of zinc, 16.9 mg/g of chromium and

17.4 mg/g of copper. Moreover, removal of pesticides oxadixyl from an aqueous solution

by using a very similar biomaterial that contains chitosan and glucans were also presented

by Saiano and Ciofalo (2007). Two of the isolates related to Phomopsis sp. (NA41 and

MNA27) showed to have a better biosorption capacity of 2.37 times higher for the isolate

NA41 and 1.58 times higher for the isolate MNA27, compared to the previously reported

study by Saiano et al. (2005). Three of the isolates related to Phomopsis sp. shows to have

better biosorption capacity of 3.41 times higher (isolate NA41), 2.28 times higher

(MNA27) and 1.33 times higher ( MNA3), compared to the previously reported study by

Sim et al. (2015).

Fungal isolate NA40 which was closely related to Xylaria sp. achieved the 2nd highest

biosorption capacity of 44.86 mg/g, while the isolates that were closely related to

Nigrospora sphaerica (NA8), Diaporthe sp. (NA31, S1 and S2), had the biosorption

capacities of 30.49, 30.93, 28.67, and 16.04 mg/g, respectively (Table 3.4). The dead

biomass of the anamorph of Diaporthe, Phomopsis, have been record to be able to biosorb

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heavy metal ions, however there is no report particularly on Diaporthe. Besides that, the

biosorption of copper using dead biomass of Xylaria and Nigrospora sphaerica also have

not been reported. Therefore, we believe that this is the 1st report on the dead biomass of

the three genus (Diaporthe, Xyalaria and Nigrospora sphaerica) in the biosorption of the

heavy metal copper.

Table 3.4: Biosorption capacity (mg/g) of the Dead biomass of the isolated fungal on

heavy metal copper biosorption.

Fungal

Isolates

Biosorption of Heavy metal

copper using Dead Biomass (mg/g) Closest match

NA8 30.49 Nigrospora sphaerica strain BTN39

[KM510416]

NA25 9.87 Phomopsis sp. 45GP/T

[GQ352480]

NA27 11.05 Phomopsis sp. 45GP/T

[GQ352480]

NA28 15.42 Phomopsis sp. 45GP/T

[GQ352480]

NA31 30.93 Diaporthe sp. P051

[EF423532]

NA40 44.86 Xylaria sp. 4Y-Cs2-1

[AB741621]

NA41 59.33 Uncultured fungus

[FR863605]

S1 28.67 Diaporthe sp. 99AS/S

[GU066666]

S2 16.04 Diaporthe sp. 99AS/S

[GU066666]

MNA3 23.07 Phomopsis sp. M31

[HM595507]

MNA27 39.73 Fungal sp. mh500.1

[GQ996123]

- 75 -

Comparison between Biosorption of Copper by Fungal Live and Dead Biomass

As we can see in the Tables 3.3 and 3.4, some of the fungal isolates that belong to the

same genus, displayed very different biosorption capacities. For example, isolate NA25,

NA27 and NA28, all are closely related to Phomopsis sp., however their live and dead

biomass biosorption capacity of heavy metal copper are vary among each other

(biosorption capacity of 52.70 mg/g of live biomass and 9.87 mg/g of dead biomass for

NA25, 59.81 mg/g of live biomass and 11.05 of dead biomass for NA27, while 52.31 of

live biomass and 15.42 mg/g of dead biomass for NA28). Besides that, isolate S1 and S2

were closely related to Diaporthe sp. but recorded different biosorption capacities of

copper (Biosorption capacity of 43.89 mg/g of live biomass and 28.67 mg/g of dead

biomass for S1, while 44.53mg/g of live biomass and 16.04 mg/g of dead biomass of S2).

Similar results have been observed by Redman et al. (2011). Endophytic fungi of the same

species (Colletotrichum magna, path-1 and L2.5) were inoculated into pepper plant

(Capsicum annuum) and resulted in different drought tolerance. Biosorption capacity can

also be affected by different chemical compositions of the cell wall. Aspergillus fungus

and Mucor rouxii have been found to possess different percentages of chitin in the cell

wall, which has been linked to their differing biosorption capacities (Volesky 1990).

Interestingly, the 4 Phomopsis sp. isolated from the roadside (labelled with NA) have

higher copper biosorption capacity using live biomass compared to the other 2 Phomopsis

sp. isolated from the jungle plant (labelled with MNA). This might due to the reason

mentioned earlier, the pre-exposure and long-term adaptation of the endophytic fungi

along the roadside. Similar results also obtained from the research done by Helander

(1995) showing better tolerant of heavy metal nickel and copper in –vitro of the

endophytic fungal strain, Hormonema sp., isolated from the plant taken near the pollution

source in Harjavalta factory than those isolated from the plant taken 8 km from the factory,

where the pollution level is lower. Ge et al. (2011) suggested that the adaptation of the

fungi toward the heavy metal will also lead to the modification of the hyphae cell surface

which is believed to be involved in fungal intracellular detoxification of the heavy metal.

Based on our results, there is a clear distinction between absorption capacities if living or

dead biomass is used. Eighty percent of the roadside isolates displayed higher copper

- 76 -

biosorption capacity using living biomass, whereas none of the jungle isolates displayed

the same trend. However, when using dead biomass, no trend was observable between

roadside or jungle isolates. A reason might be that the biosorption of heavy metals using

dead biomass is metabolism independent, and therefore purely depends on physical

adsorption (Aksu et al. 1992, Kuyucak & Volesky 1988b), ion exchange (Muraleedharan

& Venkobachar 1990; Kuyucak & Volesky 1988) and complexation (Aksu et al. 1992).

Dead biomass is usually preferred for metal biosorption due to better biosorption

efficiency compared to living biomass. However, our research shows that living biomass

has a higher biosorption capacity compared to dead biomass. Similar results were

obtained by Abedin (2014), where the living biomass of Penicillium oxalicum JQ624873

had a higher Cu biosorption efficiency of 59%, compared to 48% using dead biomass

Another study by Kahraman et al. (2005) also found that living biomass of two white rot

fungi and Phanerochaete chrysosporium had higher Cu adsorption capacity compared to

the dried biomass. Kapoor et al. (1998) achieved significant improvements in Cu

biosorption when the live fungal biomass of Aspergillus niger was pre-treated with

formaldehyde, dimethyl sulphoxide and sodium hydroxide. However, such pre-treatment

has not been undertaken in this study, which underlines the capability of our isolates to

actively deal with high Cu concentrations and also opens possible routes of further

maximising their biosorption capacities.

3.4 Conclusion

In total, 147 fungal isolates were collected 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. Only 7.5% of the total isolates managed to survive in copper concentrations up

to 1000 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.) exhibited the highest Cu biosorption capacity using dead biomass (73.26 mg/g).

This is the first time that the heavy metal 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

- 77 -

were studied. Our studies highlighted that fungal biosorption capacity is highly dependent

on the sampling area (roadside vs. jungle) and the fungal species. It also highlighted that

different biosorption mechanisms (live- metabolic dependent and dead biomass-

metabolic independent) result in different amounts of copper being removed from the

solutions.

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Chapter 4

Proteomics analysis of the Nepenthes

ampullaria associated endophytic fungus,

Xylaria sp.

Changi Wong1*, Daniel Tan1, Samuel Lihan2, Aazani Mujahid2, Jameel R. Al-Obaidi3,

Norasfaliza Rahmad3, 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

Kota Samarahan, Sarawak, Malaysia.

3 Agro-Biotechnology Malaysia Institutes, c/o MARDI Headquarters, Serdang 43400,

Selangor, Malaysia.

*Corresponding author

Email: cgwong@swinburne.edu.my

Phone number: +60168716911

ABSTRACT

Proteomics study is one of the ways to study the differentially expressed proteins of the

endophytic fungus in response to heavy metal. In this study, the previously reported

Nepenthes ampullaria associated endophytic fungus, Xylaria sp., was used to perform

proteomics analysis in heavy metal copper exposure. In total, 11 proteins spots were

found to be up-regulated and 1 protein spots was found to be down-regulated in response

to heavy metal copper. The protein spots were identified to be related to the enzymes that

involve in heat shock protein, DNA repairing and antioxidant catalysation. To our

knownledge, this study is the first study on Xylaria and serves as a base line study for the

response of this particular fungus genus to the heavy metal copper.

Keywords: Endophytic fungi, Proteomics, Heavy metal copper, Xylaria, Nepenthes ampullaria

- 79 -

4.1 Introduction

Endophytes are microorganisms (bacteria (Hallmann et al. 1997) and fungi (Araújo et al.

2001; Hallmann et al. 1997) that live symbiotically and asymptomatically within their host

plants tissue. The host plant provides nutriments and restful habitation for them, while in

return they will protect their host plant from biotic and abiotic stress such as drought and

salinity resistance (Tadych & White 2009; Rodriguez et al. 2008), thermotolerance

(Redman et al. 2002), improved plant nutrients uptake and heavy metal tolerance (Tadych

& White 2009). Endophytic fungi are known to produce valuable bioactive compounds

and enzymes that exhibit anti-microbial (Li et al. 2000; Li et al. 2001; Miller et al. 1998),

anti-cancer (Li et al. 1996), immunosuppressant (Lee et al. 1995), pectinase, laccase, and

protease properties (Maria et al. 2005). Other than mutualism with the host plant,

endophytic fungi itself are known to be capable to resist certain toxic heavy metals such

as cadmium (Deng et al. 2014; El-Gendy et al. 2011), zinc (Deng et al. 2014) and copper

(El-Gendy et al. 2011), and bioaccumulate or biosorb them (Deng et al. 2014; El-Gendy

et al. 2011). For example, Phomopsis sp. NA41, an endophytic fungi isolated from

Nepenthes ampullaria, was found to be able to tolerate heavy metal copper concentrations

up to 1000 ppm and to remove the heavy metal from solution by using its live and dead

biomass (Wong et al. submitted). Besides that, fungi are also known to increase or

decrease the expression of certain proteins under heavy metal stress. For instance,

fourteen up-regulated and 21 down-regulated proteins were detected from Phanerochaete

chrysosporium under lead stress (Yıldırım et al. 2011), and when the particular fungal

strain underwent copper and cadmium stress, 74 copper upregulated proteins and 80

cadmium upregulated proteins spots were detected (Ozcan et al. 2007).

Heavy metal copper is one of the essential micronutrients required for the cell to carry

out biochemical processes. However, excess in copper may be toxic to the cell due to the

oxidative potential of the copper towards the proteins (Letelier et al. 2005), lipids (Zhao

et al. 2014) and nucleic acids (Linder 2012), leading to lethal microbial cell damage. In

order to survive the heavy metal induced oxidative stress, microorganisms such as yeast

(Jo et al. 2008) and fungi (Yıldırım et al. 2011), were found to increase the production of

the enzymes that involve in cell protecting such as antioxidant (Yıldırım et al. 2011, Rout

& Sahoo 2013), DNA repair (Jo et al. 2008; Yıldırım et al. 2011), and heat shock protein

(Yıldırım et al. 2011). According to González-Guerrero et al. (2010), GintABC1, which

- 80 -

encodes a putative ABC transporter of the MRP subfamily, was induced by copper and

cadmium stress in Glomus intraradices. Besides that, Pimt1 genes, which encode for the

metallothioneins that are involved in metal tolerance of most eukaryotes, were detected

in Paxillus involutus by Bellion et al. (2007). While it is possible to detect most of the

genes related to heavy metal resistance using genomic studies, the biological mechanisms

such as protein expression and protein-protein interaction, cannot be explored using

genomic studies, but by proteomics.

Proteomics, the study of the proteome, can provide information on the structure and

function of a protein which we cannot obtain using genomic studies. Proteomes are

dynamic, in contrast to the relatively static genomes. Proteomes often undergo changes

in response to extra- and intracellular signals and has been defined as “the proteins present

in one sample (tissue, organism, and cell culture) at a certain point in time” (Rastogi et al.

2006; Ravi et al. 2013).

Xylaria, under the family of Xylariaceae, is a fungal genus that has a wide geographical

distribution (Patil et al. 2012; Stadler et al. 2014). It has been found as wood decaying

fungi and associated with termite nests (Ju & Hsieh 2007). Besides that, Bayman et al.

(1998) and Ratnaweera et al. (2014) also showed that Xylaria exist as endophytic fungi

in certain plants such as asubo and Australian pine and orchid. Xylaria are previously

famous for their antimicrobial properties (Ratnaweera et al. 2014), cytotoxic properties

(Inthe et al. 2014) and wide enzymatic properties (Wei et al. 1992; Liers et al. 2007; Liers

et al. 2006). However, the study of its proteome expression towards copper has yet to be

explored and recorded.

In a previous study (Wong et al., submitted), an endophytic fungi isolate related to Xylaria

sp. exhibited the ability to survive copper concentrations up to 1000 ppm. It also displayed

the highest biosorption capability (using its living biomass) among all the others 11 fungal

isolates, including Phomopsis, Diaporthe, and Nigrospora. Therefore, it was chosen to

identify differentially expressed proteins in response to treatments with 3 different

concentration of copper (0, 300, and 500 ppm). This study is the first to our knowledge

- 81 -

on Xylaria and serves as a base line study for the response of this particular fungus genus

to the heavy metal copper.

4.2 Methodology

4.2.1 Culture Conditions

The fungus (NA40) was cultured in Potato Dextrose Broth (PDB) supplied with 2

different concentrations of copper (300 and 500 ppm) and another PDB without copper

as control for 3 weeks at 25oC. After 3 weeks incubation, the fungal biomass was

harvested and underwent protein extraction. All conditions were performed in triplicates.

4.2.2 Protein Extraction

The TCA-acetone extraction performed by Méchin et al. (2007) was used to extract the

protein from the fungal cells. 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 1 g of the powdered fungal biomass with 1.8 ml of 10%

trichloroacetic acid in cold acetone containing 0.07% β-mercaptoethanol and vortexed at

4oC. After that, the mixture was incubated in -20oC overnight and on the following 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 rinsing solution (each of the 1 g with 1.8 ml

rinsing solution) containing 0.07% β-mercaptoethanol in cold acetone. The solution was

then incubated at -20°C for 1 h (mixed at 15 min intervals) and again centrifuged at

10,000g at 4oC for 15 minutes. The supernatant was discarded and this rinsing step

repeated twice. The pellet was then vacuum-dried and re-suspended with lysis buffer. The

supernatant was collected and stored at -80oC. The protein concentration was determined

using Biorad Bradford Reagent assay.

4.2.3 2-DE and Image Analysis of Protein Spots

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 samples. IEF was

- 82 -

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). 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). The gels were stained with silver staining

(Shevchenko et al., 1996).

4.2.4 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. 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.

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.

- 83 -

4.3 Results and Discussion

Proteome analyses of fungi in response to the heavy metal copper have been widely

studied using different types of fungi (Cherrad et al. 2012; Yıldırım et al. 2011). However,

no proteomic study of Xylaria sp. in response to copper has been published so far. The

endophytic fungi used in this study -NA40 (closely related to Xylaria sp. – identified

through molecular and morphological methods; see Wong et al., submitted)- was isolated

from Nepenthes ampullaria collected from the roadside of Kota Samarahan, Kuching,

Sarawak, Malaysia. In the following, we discuss its proteome response in the exposure

with varying levels of copper.

Effect on Copper treated on Fungal isolate NA40, Xylaria sp.

The fungus was exposed to two levels of copper (300 and 500 ppm) for three weeks before

protein extraction. After 3 weeks of incubation, the growth rate of the fungus was

significantly reduced in comparison to the copper-free control (data not shown). A total

of 12 protein spots were identified (see Table 4.1) with the 11 spots been up-regulated

and 1 spot been down-regulated (see Table 4.2 and 4.3). Some of the proteins have not

yet been recorded in heavy metal copper stress, while some of the proteins were known

to be heat shock protein, which are expressed under normal conditions and are highly

expressed under stress conditions such as oxidative stress (Becker & Craig 1994), viral

infections (Becker & Craig 1994; Valle et al. 2005) and elevated temperature (Becker &

Craig 1994).

Upregulated Protein spots

As can be seen in Figure 4.1, Formamidopyrimidine-DNA glycosylase is up-regulated by

the fungus in response to copper in order to repair DNA damage. Formamidopyrimidine-

DNA glycosylase is involved in repairing damaged DNA caused by oxidative stress

(UniProta). A study conducted by Boiteux et al. (1992) shows that,

Formamidopyrimidine-DNA glycosylase protein is able to specifically recognise 8-

hydroxypurines within the DNA and repair the damaged DNA in complement with

pyrimidine-specific enzymes, in vivo.

- 84 -

Ribosome recycling factor (RRF) was observed to be up-regulated by 7.39 fold in 300

ppm copper and 3.89 fold in 500 ppm copper (Table 4.2). RRF was previously recorded

in the bacterium Brucella melitensis as a heat shock protein (Teixeira-Gomes et al. 2000).

Besides that, RRF was also reported in plant (maize leaf) responses under chromium

stress (Wang et al. 2013). To the best of our knowledge, this is the 1st report of the

involvement of RRF in a fungus (Xyalaria sp.) in response to copper stress.

A newly described pantothenate kinase - Type III pantothenate kinase (CoaX), which

belongs to the acetate and sugar kinase/heat shock protein 70 /actin (ASKHA) protein

superfamily (Yang et al. 2006) was found to be up-regulated (see Table 4.1). So far,

pantothenate kinase have only been recorded in bacteria such as Helicobacter pylori,

Mycobacterium tuberculosis, Pseudomonas aeruginosa and Bordetella pertussis (Yang

et al. 2006; Awasthy 2010). To the best of our knowledge, there is so far no record of a

fungal strain producing this type of pantothenate kinase (CoaX). CoaX is known to be

involved in the catalyzation of the 1st step in the coenzyme A biosynthesis and is known

to be different from previously reported Type I and Type II pantothenate kinase proteins.

Unlike the Type I and Type II pantothenate kinase, CoaX is not inhibited by CoA and

thioesters. Nicely et al. (2007) suggested that CoaX is involved in maintaining higher

intracellular Coenzyme A levels in many bacteria in order to accommodate additional

functions for the coenzyme in thiol/disulphide redox homeostasis- a homeostasis that is

important for cellular defence against oxidative stress (Hansen et al. 2009). In order to

protect the fungus from copper oxidative stress, the CoaX was found to be up-regulated

in the fungal isolate NA40 (see Table 4.2/Figure 4.1).

Adenylosuccunate synthetase (purA) was previously reported by Winter et al. (2005) to

be up-regulated in Escherichia coli (ΔrpoH strains) when exposed to heat shock and

oxidative stress. The fungal isolate NA40 shows similar behaviour, purA was up-

regulated 6.31 fold when exposed to 300ppm copper and 1.93 fold when expose to

500ppm copper (Table 4.2/ Figure 4.1). purA is involved in the production of fumarate,

an important component of the tricarboxylic acid cycle (TCA) that yields high amounts

of NADH and ATP (Honzatko & Fromm 1999; Horecker & Stadtman 2014). The up-

regulation of the purA in the fungal isolate NA40 suggests that the protein is necessary

to provide energy for the fungal cell to reduce the oxidative damage by the copper, and

also for the metabolic changes that occur in the cell under oxidative stress.

- 85 -

Ketol-acid reductoisomerase has previously been recorded in response to heat stress

(Ferreira et al. 2006), cold shock (Graumann et al. 1996) and salt stress (Zhou et al. 2011),

and is known to be involved in the biosynthesis of amino acids (valine, leucine and

isoleucine) and catalysation of the following two processes (UniProtb):

1. (R)-2,3-dihydroxy-3-methylbutanoate + NADP+ = (S)-2-hydroxy-2-methyl-3-

oxobutanoate + NADPH

2. (2R,3R)-2,3-dihydroxy-3-methylpentanoate + NADP+ = (S)-2-hydroxy-2-ethyl-

3 oxobutanoate + NADPH.

Reduced Nicotinamide Adenine Dinucleotide (NADPH) is an important component in

the cellular antioxidation system and source for reductive synthesis of fatty acid, steroids

and DNA (Pollak et al. 2007). Besides that, it has been record to be involved in the

syntheses of flavonoid, NADPH oxidase and lignin in plant defense (Casati et al. 1999;

Torres 2006; Ying 2008). Ketol-acid reductoisomerase was up-regulated in the fungal

isolate NA40 when exposed to copper, likely leading to an increasing production of

NADPH, flavonoid and the amino acids in order to protect the cell from oxidative

damages and repair damaged DNA.

Cyanide is a toxic chemical that can be produced, metabolized and excreted by algae,

bacteria, insects, plants, and fungi (Cipollone et al. 2008). It can be catalysed by

thiosulfate sulfurtransferase to form the less toxic form of thiocyanate. Thiocyanate has

been previously reported to exhibit roles in the hosts’ defence (Chandler & Day 2012)

and also displayed antioxidant properties in humans (Xu et al. 2009; Chandler & Day 2012).

In order to protect the cell from copper oxidative stress, thiosulfate sulfurtransferase

concentration was up-regulated in the fungal cell at 300 ppm copper and even more at

500 ppm. Similar results (up-regulation of thiosulfate sulfurtransferase) were observed in

Mycobacterium tuberculosis in response to copper (Ward et al. 2008).

Two different types of ribosomal RNA methyltransferase were detected to be up-

regulated in response to the copper stress. One was ribosomal RNA large subunit

methyltarnseferase E (rlmE), which is similar to the heat shock proteins ftsJ and rrmJ

- 86 -

(UniProtc). In recent studies, Caldas et al. (2000) and Toh & Mankin (2008) have

demonstrated that a lack of rlmE decreases growth rate and makes the mutant strain more

sensitive to antibiotics (clindamycin, lincomycin, sparsomycin and hygromycin A)

compared to the wild-type strain. Besides that, Caldas and his research team (2000) also

suggested that the methylation of Um(2552) catalyzed by RrmJ in 23S RNA not only

improves the cell growth rates, it also increases protein synthesis activity, and strengthens

ribosomal subunit interactions even at non-heat shock temperatures. In our study, rlmE

was up-regulated to protect the fungal cell from copper oxidative stress. Another type of

ribosomal RNA methyltransferase detected was ribosomal RNA small subunit

methyltransferase G (rsmG), which was recorded to be involved in streptomycin

resistance and ribosomal functioning (Okamoto et al. 2007). It is known to methylate 16S

rRNA at the N7 position of the G527 nucleotide (Okamoto et al. 2007).

The up-regulation of the proteome rsmG that yields the production of S-adenosyl-L

homocysteine is believed to be involved in cysteine synthesis (UniProtd). This idea is

supported by the up-regulation of pyridoxamine 5' -phosphate oxidase homolog, which

catalyses the oxidation of either pyridoxamine 5' -phosphate (PMP) or pyridoxine 5' -

phosphate (PNP), to form pyridoxal 5' -phosphate (PLP) (Salvo et al. 2003)- an active

form of vitamin B6 that participates in the biosynthesis of the antioxidant compound

cysteine (Schnell et al. 2014). Besides that, vitamin B6 itself also serves as an important

cofactor of many enzymatic reactions such as racemization, transamination,

decarboxylation, trans-sulfuration and deamination (Mittenhube 2001). A few studies

have unravelled the antioxidant properties of vitamin B6 (Ehrenshaft et al. 1999, Osmani

et al. 1999). For instance, vitamin B6 acts as a potential antioxidant involved in the

resistance of Cercospora nicotianae (filamentous fungus) to its own product, cercosporin,

which is a strong photosensitizer of singlet molecular oxygen (1O2) (Bilski et al. 2000).

Besides that, vitamin B6 produced by the transcriptional regulation of GintPDX1 exhibits

antioxidant properties and acts as a modulator of the reactive oxygen species (ROS) of

Glomus intraradices (arbuscular mycorrhizal fungus) (Benabdellah et al. (2009).

Benabdellah et al. (2009) also demonstrated that the transcription of similar genes

(GintPDX1) -encoding for pyridoxal 5'-phosphate biosynthesis enzymes- was found up-

regulated in responses to copper-induced oxidative damage in arbuscular mycorrhizal

fungus, Glomus intraradices.

- 87 -

TtRNA modification GTPase (MnmE), also known as TrmE, can be found in both

prokaryotes and eukaryotes and has been labelled as MSS1 (orthologs of MnmE) in

Saccharomyces cerevisiae (Meyer et al. 2009) and Aspergillus clavatus (NCBIa). MnmE

(termed MSS1) catalyse the biosynthesis of the hyper-modified nucleoside 5-

methylaminomethyl-2-thiouridine (mnm5s2U34) (Elseviers et al. 1984), which is present

at the wobble position (position 34) of the specific tRNAs for lysine and glutamic acid

(Krüger & Sørensen 1998). It was found up-regulated by the fungal isolate NA40 in

response to heavy metal copper stress. MnmE (termed MSS1) have been previously

recorded to be involved in rapid growth at unfavourable conditions (Singh et al. 2009)

and stress responses such as low temperature in psychrophile Pseudomonas syringae

(Singh et al. 2009) and low pH resistance in Escherischia coli by regulating glutamate-

dependent acid resistance (Gong et al. 2004). It seems that they play a similar role for the

fungal isolate NA40.

Uncharacterized protein C24B10,16c is currently known to be an orphan gene (a gene

with no homologues of other organisms in genomes). The protein is upregulated when

exposed to 300 ppm of copper, however, down regulation of the protein was observed

when exposed to 500 ppm copper. The specific function of this protein remains poorly

understood, however, it is believed to be involved in meiotic cell cycle and proteasome

assembly (UniProte).

Down regulated protein spot

Down regulation of the protein Cell division protein SepF was observed in response to

copper (Table 4.3/ Figure 4.2). The protein SepF is involved in late cell division

(UniProtf). This can be an indication that the cell division rate of the fungal isolate NA40

was reduced when exposed to copper (observation supported based on visual inspection

of the fungal biomass; data not shown). Khan and Lee (2013) also demonstrated that

fungal growth rates will reduce in response to heavy metals such as copper and cadmium.

- 88 -

Table 4.1: List of identified proteins produced (upregulated and downregulated) in response of heavy metal copper.

Spot no /ID

Top Ranked Protein Name

[Species] Accession No. Protein

MW Protein

PI Pep.

Count Protein Score

2 Ribosome recycling factor RRF_SYNPW 20509 5.5 7 60

3 Type III

pantothenate kinase

COAX_PELUB 27859 10.8 10 81

4

Pyridoxamine 5' -phosphate

oxidase homolog

YL456_YEAST 23450 5.8 6 72

9 Uncharacterized

protein C24B10,16c

YJNG_SCHPO 13546 5.1 6 64

16 Adenylosuccunate synthetase PURA_HELAH 46631 6.1 7 63

30

Ribosomal RNA small

subunit methyltransfera

se G

RSMG_BORBZ 24149 10 9 59

33 Cell division protein SepF SEP_MYCLB 24001 6 11 72

36 Formamidopyri

midine-DNA glycosylase

FPG_VIBRA 30362 9.2 5 63

49 TtRNA

modification GTPase MnmE

MNME_VIBHB 49338 4.6 7 62

55 Ketol-acid

reductoisomerase

ILVC_PROM0 36610 5.2 7 63

67

Thiosulfate sulfurtransferas

e RDL2,MITOC

ONTRIAL

RDL2_YEAST 16744 10.1 7 53

82

Ribosomal RNA large

subunit methyltarnsefer

ase E

RLME_HALLT 29364 4.1 6 61

- 89 -

Table 4.2: Up-regulated proteins in response to copper.

Spot

Number Protein name Accession number

Treatment/Control

(ratio)

300ppm 500ppm

36 Formamidopyrimidine-DNA

glycosylase FPG_VIBPA 1.77 3.17

2 Ribosome recycling factor RRF_SYNPW 7.39 3.89

3 Protein: Type III pantothenate

kinase COAX_PELUB 3.56 1.58

4 Pyridoxamine 5' -phosphate

oxidase homolog YL456_YEAST 2.63 1.71

82 Ribosomal RNA large subunit

methyltarnseferase E RLME_HALLT 1.52 3.08

16 Adenylosuccunate synthetase PURA_HELAH 6.31 1.93

30 Ribosomal RNA small subunit

methyltransferase G RSMG_BORBZ 3.42 1.39

67 Thiosulfate sulfurtransferase

RDL2,MITOCONTRIAL RDL2_YEAST 2.00 2.77

49 TtRNA modification GTPase

MnmE MNME_VIBHB 5.52 2.39

55 Ketol-acid reductoisomerase ILVC_PROM0 1.20 2.45

9 Uncharacterized protein

C24B10,16c YJNG_SCHPO 2.99 0.92

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

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Figure 4.1: Up-regulated protein spots in response to copper.

Figure 4.2: Down-regulated protein spot in response to copper.

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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.

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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

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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).

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