STUDY AND ANALYSIS ON HYDROCARBON BIOREMEDIATION POTENTIAL

OF DIFFERENT FUNGAL GENERA

MOHD FARITH BIN KOTA

A thesis submitted

In fulfillment of the requirements for the degree of Master of Science

Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2013

ii

Acknowledgement

In the name of God, the Most Gracious, the Most Merciful,

First of all, I would like to thank Him, for His grace and mighty in giving me the strength and

wisdom to complete this thesis. I wish to express my sincere thanks to my supervisor and co-

supervisor, Associate Professor Dr. Awang Ahmad Sallehin Awang Hussani and Dr. Azham

Zulkharnain for their dedication, patience and guidance in the completion of my study. I also

thank my family for their unceasing encouragement and support.

I place on record, my sincere gratitude to Dr. Micky Vincent and Associate Prof. Dr. Mohamad

Suffian for their expert, sincere and valuable comments in improving this thesis. I would like

to thank, the Ministry of Higher Education Malaysia (MoHE), Universiti Malaysia Sarawak

and Centre of Excellence in Sago Research (CoESAR), Universiti Malaysia Sarawak for the

financial support during the course of this research.

I take this opportunity to record my sincere thanks to all the department members of

Department of Molecular Biology, especially to the all lab members of Molecular Genetic Lab,

for their help and encouragement. Last but not least, my senses of gratitude to one and all,

directly or indirectly, have lent their helping hand in this research.

iii

STUDY AND ANALYSIS ON HYDROCARBON BIOREMEDIATION POTENTIAL

OF DIFFERENT FUNGAL GENERA

MOHD FARITH BIN KOTA

Resource Biotechnology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

One of the major environmental problems today is hydrocarbon contamination from the activities related to the

petrochemical industry. Bioremediation is a promising technology for the treatment of these contaminated sites as

it is cost effective and lead to complete mineralization. This research attempts to study the potential of different

fungal genera for potential use in bioremediation of hydrocarbon. Aspergillus flavus UMAS HDF8, Aspergillus

versicolor UMAS HDF6, Bionectria ochroleuca UMAS BHDF7, Penicilium chermisinum UMAS HDF2 and

Trichoderma virens UMAS HSF7 were employed for the bioremediation purposes. Screening of fungal strain

sensitivity towards hydrocarbons was first conducted. In order to enhance the growth of fungi on hydrocarbon

contaminated soil, the suitable bulking agent was selected. The hydrocarbon degradation trial was conducted for a

period of six weeks, followed by post-treatment tests. All of the fungal strains showed high tolerance towards

hydrocarbon. Sago waste (sago hampas) is found as the most suitable bulking agent as all of the fungal test strains

capable to growing on it. A significant difference was found in the ability of B. ochroleuca UMAS BHDF7 to

degrade hydrocarbon. B. ochroleuca UMAS BHDF7 was able to degrade more than 80% of the C12 to C28, with

100% degradation of C12 and C28.

Keywords: hydrocarbon, bioremediation, fungi, sago waste, Bionectria ochroleuca.

ABSTRAK

Pencemaran hidrokarbon daripada aktiviti berkaitan dengan industri petroleum merupakan salah satu punca

masalah alam sekitar yang kian meruncing. Bioremediasi merupakan teknologi yang sesuai bagi memulihkan

tapak yang telah tercemar kerana ianya efektif dari segi kos dan membolehkan penguraian berlaku dengan

lengkap. Kajian ini cuba untuk mengkaji potensi di kalangan kulat tempatan pengurai hidrokarbon dari genera

yang berbeza yang telah berjaya dipencilkan. Aspergillus flavus, Aspergillus versicolor, Bionectria ochroleuca,

Penicilium chermisinum dan Trichoderma virens digunakan bagi tujuan bioremediasi hidrokarbon. Pemilihan

kulat adalah berdasarkan kepada tahap sensitiviti kulat terhadap hidrokarbon yang perlu dijalan terlebih dahulu.

Agen pemukal dipilih bagi memastikan pertumbuhan kulat yang baik. Ujikaji penguraian hidrokarbon dijalankan

untuk tempoh selama enam minggu,diikuti dengan beberapa ujian pasca-penguraian. Keputusan analisa

menunjukkan semua kulat yang dikaji mempunyai tahap toleransi yang tinggi terhadap hidrokarbon. Hampas

sagu merupakan agen pemukal yang terbaik kerana semua kulat mampu tumbuh dengan baik di atasnya.

Perbezaan ketara ditunjukkan oleh Bionectria ochroleua bagi penguraian hidrokarbon. Bionectria ochroleuca

menunjukkan kadar penguraian lebih daripada 80% untuk karbon-12 hingga karbon-28, dengan penguraian yang

lengkap bagi karbon-12 dan karbon-28.

Kata kunci: hidrokarbon, bioremediasi, kulat, sisa sagu, Bionectria ochroleuca

iv

TABLE OF CONTENTS

Acknowledgement

Abstract

ii

iii

Table of Contents iv

List of Tables vi

List of Figures vii

List of Abbreviations ix

1.0 Introduction and Objectives 1

2.0

Literature Review 4

2.1 Petroleum 4

2.1.1 Composition of petroleum hydrocarbons 4

2.1.2 Methods of analysis of petroleum hydrocarbons 5

2.1.3 Petroleum hydrocarbon contamination 6

2.2 Fungi 10

2.2.1 Fungi in the environment 10

2.2.2 Fungal decay mechanisms 11

2.2.3 Lignin modifying enzymes 13

2.2.4 Fungi in bioremediation 15

2.3 Bioremediation of contaminated soil by fungi 17

3.0

Materials and Methods 19

3.1 Microbial inoculums preparation 19

3.2 Liquid growth medium, buffer solution and soil preparation 19

3.3 Screening of fungi strains sensitivity towards hydrocarbons 20

3.4 Bulking agent 21

3.5 Fungal growth study in artificially contaminated soil 21

3.6 Screening of fungi for bioremediation of hydrocarbon contaminated soil 22

3.7 Bioremediation of hydrocarbon contaminated soil 23

3.7.1 Hydrocarbon analysis 24

3.7.1.1 Soxhlet extraction 24

3.7.1.2 Cold toluene 24

v

3.7.1.3 Gravimetric analysis 24

3.7.1.4 Gas Chromatography Flame Ionization Detector

(GC/FID)

25

3.7.2 Bioremediated soil toxicity test 26

3.7.2.1 Microbiological analysis 26

3.7.2.1 Root length analysis 26

3.8 Enzyme assays 26

3.9 Optimization of crude oil bioremediation via response surface

methodology (RSM)

28

4.0

Results and Discussions 30

4.1 Screening of fungi strains sensitivity towards hydrocarbons 30

4.2 Bulking agent 31

4.3 Fungal growth study in artificially hydrocarbon contaminated soil 34

4.4 Screening of fungi for bioremediation of hydrocarbon contaminated soil 37

4.5 Bioremediation of hydrocarbon contaminated soil 39

4.6 Enzyme assays 45

4.7 Optimization of crude oil bioremediation by B. ochroleuca via response

surface methodology (RSM)

47

4.7.1 Full model regression and statistical analysis 48

4.7.2 Reduced model regression and statistical analysis 52

4.7.3 Correlation among variables 54

4.7.4 Numerical optimization and verification 57

5.0 Conclusion 59

6.0 Recommendation 60

8.0 References 61

Appendix A: Sterilization and Media

81

Appendix B: Raw Data 83

Appendix C: List of Original Publications

vi

LIST OF TABLES

Table Descriptions Page

Table 1 Major oil-spill at international level 9

Table 2 The list of indigenous fungi species used 19

Table 3 Parameters with values for crude oil bioremediation by B.

ochroleuca 29

Table 4 Results for visual observation of mycelia extension on

contaminated PDA plate 30

Table 5 Results for visual observation of fungal growth of different

bulking agents 34

Table 6 Percentage of hydrocarbon degradation for week 6 for all species

with sterile and unsterile control 39

Table 7 Gravimetric analysis for each fraction of crude oil extracted from

contaminated soils on Week 6 41

Table 8 Number of bacteria colonies on Nutrient Agar after 24 hours 43

Table 9 Number of fungi growth on Potato Dextrose Agar 44

Table 10 Root length analysis (n=10) 45

Table 11 Analysis of variance for the regression model and respective

model terms 51

Table 12 Summary statistics of the full model 52

Table 13 Parameters and values for crude oil bioremediation (reduced

model) 53

Table 14 Analysis of variance for the regression model and respective

model terms 54

Table 15 Summary statistics of the reduced model 55

Table 16 Optimized combination variables for hydrocarbon degradation 58

Table 17 Numerical optimization and verification of optimized combination

variables for hydrocarbon degradation 59

vii

LIST OF FIGURES

Figure Descriptions Page

Figure 1 Bay and fjord regions in PAH molecular structure. 7

Figure 2 Generalized pathways of PAHs metabolism by fungi and bacteria

(adapted from Cerniglia, 1993).

16

Figure 3 Fungi growth on agar amended with crude oil. (a) P. chermesinum,

(b) A. versicolor, (c) T. virens, (d) A. flavus, and (e) B. ochroleuca.

30

Figure 4 B. ochroleuca growth on different bulking agent on day 14. (a)

Mixture of all bulking agents, (b) Oil palm empty fruit bunch, (c)

Rice husk, (d) Sawdust, and (e) Sago waste (Sago hampas).

32

Figure 5 P. chremesinum growth on different bulking agent on day 14. (a)

Mixture of all bulking agents, (b) Oil palm empty fruit bunch, (c)

Rice husk, (d) Sawdust, and (e) Sago waste (Sago hampas).

32

Figure 6 A. versicolor growth on different bulking agent on day 14. (a)

Mixture of all bulking agents, (b) Oil palm empty fruit bunch, (c)

Rice husk, (d) Sawdust, and (e) Sago waste (Sago hampas).

32

Figure 7 T. virens growth on different bulking agent on day 14(a) Mixture of

all bulking agents, (b) Oil palm empty fruit bunch, (c) Rice husk,

(d) Sawdust, and (e) Sago waste (Sago hampas).

33

Figure 8 A. flavus growth on different bulking agent on day 14. (a) Mixture

of all bulking agents, (b) Oil palm empty fruit bunch, (c) Rice husk,

(d) Sawdust, and (e) Sago waste (Sago hampas).

33

Figure 9 B. ochroleuca growth on soil with addition of sago waste.

Microscopic picture were taken through microscope with

magnification power of 40X.

35

Figure 10 P. chremesinum growth on soil with addition of sago waste.

Microscopic picture were taken through microscope with

magnification power of 40X.

36

Figure 11 A. versicolor growth on soil with addition of sago waste.

Microscopic picture were taken through microscope with

magnification power of 40X.

36

Figure 12 T. virens growth on soil with addition of sago waste. Microscopic

picture were taken through microscope with magnification power

of 40X.

36

viii

Figure 13 A. flavus growth on soil with addition of sago waste. Microscopic

picture were taken through microscope with magnification power

of 40X.

37

Figure 14 Cold toluene absorbance reading at 420nm for each species. 38

Figure 15 View from top of soil spiked with hydrocarbon. (a) Sterile control.

(b) Unsterile control. (c) Soil with B. ochroleuca (replicate A). (d)

Soil with B. ochroleuca (replicate B).

40

Figure 16 Cold toluene absorbance reading at 420nm for B. ochroleuca with

sterile and unsterile control.

40

Figure 17 Percentage degradation of fraction 1 (f1) from treatment against

sterile control

42

Figure 18 Growth condition of B. ochroleuca in MSM with crude oil. 46

Figure 19 Enzyme activity for LiP, MnP, and Lac produced by B. ochroleuca

in MSM with crude oil.

46

Figure 20 Response surface 3D plot indicating the effect of interaction

between variables on crude oil degradation. (a) Temperature and

percentage of inoculums (percentage of crude oil = 8.75%). (b)

Temperature and percentage of crude oil (percentage of inoculums

= 9%). (c) Percentage of crude oil and percentage of inoculums

(temperature = 29 °C).

57

ix

LIST OF ABBREVIATIONS

ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

ANOVA Analysis of Variance

CCD Central Composite Design

°C Degree Celsius

DCM Dichloromethane

GC Gas Chromatography

GC-MS Gas Chromatography Mass Spectrophotometry

GC-FID Gas Chromatography Flame Ionization Detector

HPLC High Performance Liquid Chromatography

GYM Glucose – Yeast Extract - Malt Extract

HBT 1-hydroxybenzotriazole

H2O2 Hydrogen Peroxide

h Hour

IR Infrared Spectroscopy

ITS Internal Transcribed Spacers

kDa Kilodalton

Lac Laccase

lac genes Laccase genes

LiP Lignin Peroxidase

LDF Litter Decomposing Fungi

MnP Manganese Peroxidase

mg/l Milligram per liter

mL Milliliter

mM Millimolar

MSA Minimal Salt Agar

nm Nanometer

OPEFB Oil Palm Empty Fruit Bunch

% Percent

PAHs Polycyclic Aromatic Hydrocarbons

PCR Polymerase Chain Reaction

PDA Potato Dextrose Agar

rpm Revolution Per Minute

s Seconds

SOM Soil Organic Matter

TLC Thin Layer Chromatography

VA Vertaryl Alcohol

VP Versatile Peroxide

v/w Volume over Weight

WRF White Rot Fungi

1

1.0 Introduction

Demand for petroleum as a source of energy and as the primary raw material for the chemical

industry has increased each year. In 2011, 600,000 barrels of crude oil were produced each

day (Ministry of Finance Malaysia, 2011). High production efficiency requires cautious

handling procedures. There has been a growing public concern regarding the release of crude

oil into the environment. Crude oil is known to enter the environment frequently in large

volume via several means. One of the major and natural routes is the seepage from natural

deposits that leads to the appearance of crude oil in marine environments. Apart from that,

crude oil may enter the environment due to accidental release during production, storage, and

transportation. Recent example can be seen from the explosion on Deepwater Horizon oil rig

in the Gulf of Mexico. On 20 April 2010, explosion on the oil rig resulted in eleven crew

members’ fatalities and approximately 4.9 million barrels of crude oil was released into the

environment over the period of 84 days from the explosion (BP, 2010).

Malaysia has been developing deep-water fields, rejuvenating old areas and

introducing incentives to develop less-profitable fields in a bid to increase the country's

output as global energy use climbed. Oil refineries major hitch is the safe disposal of oil

sludge. A typical oil refinery in Malaysia which is capable of producing 105,000 barrels per

day will produce roughly 50 tons of sludge per year (Agamuthu, 2011). Oil sludge need to be

handled carefully since its constituents are carcinogenic, immunotoxicants and mutagenic.

The toxicity profiles of petroleum hydrocarbons to microorganisms, plants, animals and

humans are well established. Incardona et al. (2005) reported that fish embryos that are

exposed to low levels of PAHs in crude oil develop a syndrome of edema and craniofacial

2

and body axis defects. Proficient technology in term of cost and efficiency is highly

demanded in order to remove or degrade the hazardous constituents of oil sludge.

Bioremediation appear to suit the characteristics of the demanded technology.

Bioremediation is a process of using microorganisms to convert hazardous pollutants into less

toxic compounds (Kirch, 2008; Odgen & Adams, 1989). Accompanied by some litter

decomposing fungi, white rot fungi (WRF) are the only organisms which have the ability to

degrade lignin completely. Due to the chemical aromatic structural resemblance of lignin and

petroleum hydrocarbons and some other environmental pollutants, ligninolytic fungi have

been regarded as the most promising candidates to degrade petroleum hydrocarbons (Husaini

et al., 2008; Radhukumar et al., 2006; Steffen et al., 2003; Pointing, 2001). White rot fungi

produce three types of enzymes which is involved in the degradation of lignin. The enzymes

are lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac) (Ohkuma et

al., 2001).

Therefore, the main objective of this research is to discover the potential of locally

isolated hydrocarbon degrading fungi in the bioremediation of hydrocarbon contaminated

soil. In order to do so, the specific aims are as follows:

1. To choose a suitable bulking agent and to determine whether the fungi are able to

grow and penetrate into the contaminated soil with or without bulking agent.

2. To select the best hydrocarbon degrading fungi that can degrade hydrocarbon in

hydrocarbon contaminated soil.

3

3. To carry out preliminary enzymatic studies of lignin modifying enzymes (LMEs)

produced by the selected fungi in liquid culture during biodegradation of

hydrocarbon.

4. To perform the bioremediation hydrocarbon contaminated soil trial with several post

bioremediation tests to evaluate its bioremediation efficiencies.

5. To conduct optimization of parameters for crude oil bioremediation using the best

fungus species.

4

2.0 Literature Review

2.1 Petroleum

2.1.1 Composition of petroleum hydrocarbons

Petroleum hydrocarbons are made of complex mixtures of compound which can be classified

into four main fractions; saturates (alkanes), aromatics, asphaltenes, and resins. The first

fraction, saturates can further be categorized into straight chain alkanes (normal alkanes),

branched alkanes (isoalkanes), and cycloalkanes (naphthenes) (De Oteyza et al., 2004; Díaz-

Ramírez et al., 2003). For aromatic fraction, it consists of volatile monoaromatic

hydrocarbons for instance benzene, toluene, and xylenes; polyaromatic hydrocarbons

(PAHs), naphthenoaromatics, and aromatic sulphur compounds such as thiophenes and

dibenzothiophenes (De Oteyza et al., 2004). The asphaltene and resins fractions consist of

polar molecules containing nitrogen, sulphur and oxygen (Buenrostro-Gonzalez et al., 2004).

Examples are phenols, fatty acids, ketones, esters, and porphyrins for ashpaltene where as

pyridines, quinolines, carbazoles, sulfoxides, and amides are for resins. In general,

ashpaltenes are large molecules that are mixed in oil in colloidal manner, while resins are

dissolved in oil as they are amorphous solids.

Factors such as the source, age, geological history, migration, and alteration of crude

oil play a part on relative distribution of these fractions. Sensitivity of hydrocarbons towards

microbial attack is as follows, ranked in the following order of decreased susceptibility: n-

alkanes > branched alkanes > low molecular weight aromatics > cyclic alkanes (Peters et

al., 2005; van Hamme et al., 2003; Perry, 1984). Based on the biodegradation rate, the

5

saturated fractions show the highest rates of biodegradation, followed by light aromatics, high

molecular weight aromatics, and polar compound revealed the lowest rates of biodegradation.

However, this pattern could not be applied to all microorganisms as certain species of

microorganism prefer specific substrate as carbon source. Elshafie et al. (2007) reported that

P. chrysogenum utilized C18 more compared to A. niger. On the other hand, A. niger utilized

C13 more compared P. chrysogenum.

2.1.2 Methods of analysis of petroleum hydrocarbons

An extensive selection of instrumental and non-instrumental techniques has been recognized

for the analysis of petroleum hydrocarbon. These include gas chromatography (GC), gas

chromatography - mass spectrometry (GC-MS) (Jeong-Dong & Choul-Gyun, 2007), gas

chromatography with flame ionization detector (GC-FID) (Peijun et al., 2002), high

performance liquid chromatography (HPLC) (Boonchan et. al., 2000), thin-layer

chromatography (TLC-FID) (Jiang et. al., 2008), infrared spectroscopy (IR) (Mullins et

al., 2006), isotope radio mass spectrometry, and gravimetric techniques (Hadibarata &

Tachibana, 2009). Of these options, the gas chromatography (GC) techniques are the most

famous among researchers who conducted the study on petroleum hydrocarbon. This is

followed by high-performance liquid chromatography. Usage of capillary column has made

capillary GC-MS as one of the most enhanced techniques as it can be used to analyze oil-

specific biomarker compounds and polycyclic hydrocarbons. Nowadays, advances in

computer and information technology have improved and optimized the accuracy and

precision of data handling capability, analytical data, and quality control.

6

2.1.3 Petroleum hydrocarbon contamination

Soil is defiant to perturbations, which is the changes in the function of a biological system,

induced by either external or internal mechanisms. This is due to the capability of the soil

itself to carry out its very own a set of “defense” mechanisms. The “defense” mechanisms

include sorption/desorption processes, biological or chemical reduction-oxidation reactions,

hydrolysis, volatization or chelation (Young & Jordan, 1995). However, just like any other

defensive system, if the threats kept on coming, it would not be able to resist it. Eight (8)

threats to soil quality have been identified by the European Commission in the

Communication of Thematic Strategy for Soil Protection in 2006. The eight (8) threats

include sealing, erosion, loss of organic matter, diminished of biodiversity, compaction,

hydrological risks, salinitisation and contamination (Commission of the European

Communities, 2006). Among the eight (8) threats, soil contamination demands the most

attention globally.

According to Jones et al. (2005), soil contamination can be categorized either as

localized or diffused contamination. Localized contamination is contributed frequently by

industrial in term of the discharge and waste, improper waste disposal, or accidental spills

during handling or transportations. Meanwhile, diffuse contamination is produced mostly by

intensive agriculture and forestry practices. The contaminants are very diverse in term of

chemical structure and properties. However, they have the characteristic in common of being

anthropogenic, recalcitrant and toxic.

7

Polycyclic aromatic hydrocarbons (PAHs) are made of two or more fused benzene

rings which sharing a pair of carbon atoms between two adjacent rings in either linear, cluster

or angular arrangements. PAHs are made of solely carbon and hydrogen atoms. Due to their

large negative resonance energy, PAHs are chemically stable at ambient temperature. In

addition, their hydrophobic structures contribute to their characteristic of insoluble in water

and highly lipophilic (Lui et al., 2005). The low molecular weight PAHs include naphthalene,

with two six-membered rings; biphenylene, acenaphthylene, acenaphthene, and fluorene,

with two six-membered rings and a four- or five-membered ring; and phenanthrene and

anthracene, with three six-membered rings. The high molecular weight PAHs include

fluoranthene, pyrene, benz[a]anthracene, and chrysene, with four rings; perylene,

benzo[a]pyrene, benzo[e]pyrene, and dibenz[a,h]anthracene with five rings; and many others

with six or more rings. PAHs that own bay or fjord regions in their molecular structure have

the potential to be carcinogenic as shown in Figure 1.

Figure 1: Bay and fjord regions in PAH molecular structure (adapted from Goldman et al., 2001).

Mattsson (2008) reported that when benzo[a]pyrene is in an organism, it will be

activated by a series of metabolic reactions which then lead to the formation of carcinogenic

metabolite known as diol epoxide, which may bind covalently to DNA, resulting in mutations

8

and consequently leading to formation of tumors. Microorganisms also affected by PAHs, for

instance, phenanthrene inhibits spore germination in some fungi (Lisowska et al., 2004).

PAHs are commonly found in coal and petroleum. Dimashki et al. (2000) and

Finlayson-Pitts and Pitts Jr. (1997) suggested that the principal origin of PAHs is from the

incomplete combustion of organic matter from various sources such as motor vehicles, coal-

fired power plants, home heating furnaces, and even forest fires. This was further supported

by Mastral and Callen (2000) which reported that whenever coal, petroleum products, wood,

urban solid wastes, or old tires are burned, PAHs are generated. Oil refineries, coal

gasification plant, steel mills and aluminum plants also contribute to the release of PAHs into

the environment. However, crude oil spills from pipelines and supertankers attract the most

attention as the impact is greater to the environment. Leakages from the pipelines and

accidents of supertankers deposited large amounts of PAHs on the soil and in the ocean.

PAHs can be found in soil, even in remote areas without any human settlements

(Johnsen & Karlson, 2007). Volcanic eruptions and forest fires are the major natural inputs of

PAHs. Studies done by Dabestani and Ivanoc (1999) and Dimashki et al. (2000) stated that

most PAHs originated from anthropogenic sources such as incomplete combustion of fossil

fuels, wood and waste, vehicle exhaust, and accidental spill during handling and

transportation of petroleum. Diffuse contamination occurs mainly via atmospheric deposition

of PAHs adsorbed to particles. Wind transports these particles to further localizations and the

PAHs adsorbed to particles are deposited directly onto the soil or indirectly through the

vegetation. Approximately 0.7 – 1 mg/m2 of PAHs are received by soil annually by air

emissions (Johnsen & Karlson, 2007; Wilcke, 2007; Wilcke, 2000). Collision of supertankers

9

at sea which resulted in accidental crude oil spills in the sea are important localized sources

of PAH contamination. Table 1 presents major oil-spill at international level. PAHs are

frequently deposited into the sediments or transported to the shorelines and other marine

ecosystems such as coastal marshes or estuaries, for example Arabian Gulf oil spill in 1991

(Zekri & Chaalal, 2005)

Table 1: Major oil-spill at international level

Source Location Date Barrels Spill

Iraq/Kuwaita

Persian Gulf February 1991 11,000,000

Exxon Valdezb

Gulf of Alaska April 1989 750,000

IXTOC I Wellc

Campeche Bay, Mexico June 1979 3,000,000

Amoco Cadizd

Brittany, France March 1978 1,604,500

Deepwater Horizone

Gulf of Mexico April 2010 4,900,000

a Cited from Hussain and Gondal, 2007.

b Cited from Bodkin et al., 2002 and Peterson et al., 2002.

c Cited from Atwood and Ferguson, 1982.

d Cited from Page et al., 1988 and Dauvin, 2000.

e Cited from Kujawinski et al., 2011.

On 20 April 2010, British Petroleum (BP) America Production Company’s leased

Deepwater Horizon oil rig in the Gulf of Mexico exploded, resulting in 11 crew member

fatalities (Kujawinski et al., 2011). It is regarded as one of the largest environmental disasters

of the decade. The explosion which caused by the uncontained release of hydrocarbon (oil

and natural gas) came along with the devastating environmental impact as it affected the

livelihoods of thousands of Gulf Coast citizens and businesses. The spill spanned

approximately for 84 days, and on 15 July 2010, responders gained control of the oil

discharge. Most recent estimations done by the United States (US) federal government’s

Flow Rate Technical Group, it is estimated more than 200 million gallons or 4.9 million

barrels of crude oil was released into the environment.

10

2.2 Fungi

2.2.1 Fungi in the environment

Soil organic matter (SOM) is vital in maintaining the chemical and physical properties of

soil, thus preserving soil quality and function. Microorganisms are primarily responsible for

soil organic matter (SOM) dynamics. However, Coleman and Wall (2007) suggested that the

role of micro-, meso-, and macro-fauna are also vital for helping microorganisms in the task

of colonizing and degrading organic matter. Bacteria are the most diverse group of

microorganisms found within soil, followed by fungi and archaea. There are more than 104

bacterial types are present in pristine soil (Torsvik & Øvreås, 2002). On the other hand, fungi

are more abundant in term of biomass as they can reach 2 - 5 tons per hectare as they are

colonizing the uppermost layer of the soil (Finlay, 2007). Up to date, the number of fungal

species in soil is not available due to the difficulties in isolating and characterizing soil fungi

by culturing techniques. Buée et al. (2009), suggested that with the new molecular studies

technique, Polymerase Chain Reaction (PCR) followed by next-generation sequencing of the

internal transcribed spacers (ITS), researchers now can identify fungi species more precisely.

According to Thorn and Lynch (2007), representatives of traditional phyla of the

fungi kingdom which are found in soil are as follows:

i) Chytridiomycota is represented by plant pathogens and parasites;

ii) Zygomycota includes parasitic and saprotrophic fungi;

iii) Glomeromycota includes arbuscular mycorrhiza-forming fungi;

iv) Ascomycota is the largest group with approximately 50,000 species and, thus, with

different ecological roles in the soil;

11

v) Basidiomycota includes wood-decaying and litter-decomposing fungi, soil-borne

pathogens of crops and forest trees.

2.2.2 Fungal decay mechanisms

Fungal decay mechanisms can be classified into three main types, white-rot, brown-rot and

soft-rot. Each type of fungal decay is caused by specific species of fungi. Together with some

litter decomposing fungi, white rot fungi (WRF) are the only organisms which have the

ability to degrade lignin completely (Levin et al., 2008; Clemente et al., 2001; Vyas et

al., 1994). Some WRF decay all of the components of the wood at the same time and for

those which are referred as selective white rot fungi WRF, degrade lignin and hemicellulose

only. Studies done by Martinez et al. (2005) and Hatakka (2001) reported that white rotten

wood has a fibrous and light appearance in general. Although WRF are commonly found on

the hardwoods of angiosperm trees, some species such as Phellinus chrysoloma and

Heterobasidion sp., are found to be growing in softwood, spruce and pine respectively

(Martínez et al., 2005; Carlile et al., 2001; Dix & Webster, 1995).

Lignin is a heterogeneous polymer which high in molecular weight. It is made of

phenylpropane subunits joined together by β-aryl ether, carbon – carbon, and other types of

bonding which generally contain three precursor aromatic alcohols including coniferyl,

sinapyl and p-courmaryl alcohol (Wei et al., 2009; Leonowicz et al., 1999). Due the chemical

resemblance of lignin and PAHs and some other environmental pollutants, ligninolytic fungi

have been regarded as the most promising candidates to degrade PAHs (Raghukumar et

al., 2006; Steffen et al., 2003). Phanerochaete chrysosporium, was the first WRF to be

12

scientifically proven to partially transforms radiolabeled benzo[a]pyrene in liquid cultures to

polar metabolites and CO2 as discovered by Bumpus et al. (1985).

Lignocellulosic material is vital for the growth of fungi in nature and also crucial in

bioremediation. This is due to the contaminated soil is normally poor in nutrients and the soil

itself is an extreme environment for some fungal species (Baldrian, 2008). The

lignocellulosic substrates used in fungal bioremediation normally are residues from

agriculture, forestry and food industry. Wheat straw, sugar cane bagasse, and corn cobs are

common examples of agricultural waste which used in bioremediation. Bennett et al. (2001)

and Sánchez (2009) reported that, sawdust, wood chips and bark are often used as substrates

in bioremediation. Substrate formulation is one important factor which will play a role in

successful fungal bioremediation (Meysami & Baheri 2003; Rhykerd et al., 1999; Leštan et

al., 1996).

In general, ligninocellulolytic fungal inoculum is prepared in liquid broth. Once the

fungi have grown sufficiently on the substrate, the inoculum is then introduced into the soil.

Simultaneously, the substrate they will serve as the carbon source and supporting material for

fungal hyphae colonization during bioremediation. The substrate may be placed directly on

the surface of soil, within the soil layers, mixed with the soil or embedded in a specific carrier

substance, which commonly composed of some wood residue which will act as a substrate

itself (Ford et al., 2007a; Ford et al., 2007b; Bennett et al., 2001; Eggen, 1999; Leštan &

Lamar, 1996; Leštan et al., 1996). It is an advantage if the substrate is resistant to

colonization by indigenous microorganisms in the soil (Leštan et al., 1996). However, due to

the high content of cellulose in the most of commonly applied substrate, it may be easy to be

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colonized by other fungal species, for example anarmorphic ascomycetes. Sánchez (2009)

reported that corn cobs which commonly used as substrates are rich in cellulose, 45% in total.

2.2.3 Lignin modifying enzymes

Lignin Modifying Enzymes (LMEs) are responsible for the degradation of lignin. LMEs are

made of lignin peroxidase (LiP; EC 1.11.1.14), manganese peroxidase (MnP; EC 1.11.13),

and laccase (Lac; EC 1.10.3.2). Apart from the three enzymes, versatile peroxidase (VP; EC

1.11.1.16), a hybrid form of MnP and LiP are also responsible for the degradation of lignin

(Baldrian, 2006; Martínez et al., 2005; Hofrichter, 2002; Hatakka, 2001).

Laccases are generally extracelullar multicopper oxidases, although intracellular

laccases have also been found in wood-decaying fungi. Fungal laccases are glycoproteins

which 60 – 70 kDa in size. It catalyze the oxidation of phenolic structures of lignin by

reducing one molecular oxygen to water (Baldrian, 2006; Hatakka, 2001). Laccase may also

oxidize non-phenolic compounds which have high redox potential such as PAHs, in the

presence of either natural mediators derived from oxidized lignin (p-coumaric acid or

syringaldehyde (Camarero et al., 2005) or synthetic mediators [ABTS (2,2´-azinobis(3

ethylthiazoline-6-sulfonate)) or 1-hydroxybenzotriazole (HBT) (Baldrian, 2006). Baldrian

(2006) reported that basidiomycetes, ascomycetes, and anamorphic ascomycetes capable of

producing different isoforms of laccases. P. chrysosporium, the most studied WRF for

bioremediation, has been discovered to be lacking laccase genes (lac genes), although WRF

are commonly found to be most active laccase producers. Therefore, on-going research is

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being conducted to understand the actual involvement of laccase in lignin degradation

(Hatakka & Hammel, 2010; Lundell et al., 2010).

Manganese peroxidases (MnP) are glycoproteins with heme, which come in the size

of 38 – 62 kDa. MnP catalyze oxidation of Mn2+

to Mn3+

using H2O2 as an electron acceptor

(Hofrichter, 2002). Mn3+

is a strong oxidant, which able to oxidize phenolic and aromatic

amines to phenoxyl and amino radicals, respectively, after chelation and stabilization with

carboxylic such as malic acid (Hofrichter, 2002; Kuan & Tien, 1993; Wariishi et al., 1992).

The catalytic cycle of MnP involves two oxidative states of enzyme; compound I (MnP oxid

I) and compound II (MnP oxid II). The catalytic cycle start with binding of H2O2 to the

native enzyme, resulting in formation of the compound I (MnP oxid I). Then, MnP oxid I will

be reduced to MnP oxid II, which will further reduced to the native enzymes. The reduction

occurs via one electron oxidation of Mn2+

to Mn3+

. Hofrichter (2002) reported that MnP

production is limited to basidiomycetes and the majority of WRF and litter decomposing

fungi (LDF) have the ability to secrete MnP.

Lignin peroxidase (LiP) is less common than MnP or laccase among WRF, and yet no

LDF have been found to produce LiP (Ruíz-Dueñas & Martínez, 2009). Lignin peroxidase

(LiP) is less common compared to MnP or laccase among WRF. Ruíz-Dueñas and Martínez

(2009) reported that no litter decomposing fungi (LDF) have the ability to produce LiP. Lip is

a glycosylated peroxides which contain heme and is 40 kDa in size. It is the only enzyme

capable of oxidizing both phenolic and non-phenolic compounds via the reduction of H2O2.

As a result from the reduction of H2O2, aryl cation radicals are formed (Hammel & Cullen,

2008; Hatakka, 2001). Veratryl alcohol (VA) is a common substrate for LiP, which may act

15

as a diffusible mediator oxidizing β-O-4 lignin dimer or non-accessible lignin substructures

(Hammel & Cullen, 2008; Hatakka, 2001).

Pe´rez-Boada et al. (2005) and Camarero et al. (1999) and proposed that versatile

peroxidase (VP), a hybrid enzyme possessing the catalytic properties of LiP and MnP, is also

one of the lignin modifying enzymes. In contrast to MnP and LiP, VP capable of oxidizing

both low and high redox potential compounds with or without the aid of Mn3+

(Ruíz-Dueñas

& Martínez, 2009). Due to this characteristic, VP is a promising enzyme with large potential

for industrial application as well as in the field of contaminant degradation (Pozdnyakova et

al., 2010). Bjerkandera and Pleurotus species are the only known species to produce VP

(Ruíz-Dueñas & Martínez, 2009; Hammel & Cullen, 2008).

2.2.4 Fungi in Bioremediation

In 1973, Cerniglia and associates published a study on the potential of a non-ligninolytic

fungus, Cunninghamella elegans to degrade crude oil (Cerniglia & Perry, 1973). It was the

first time that fungi were proposed as an agent to degrade contaminants. The same authors

then concluded that C. elegans used the same mechanism which is similar to mammals to

metabolize PAHs. This mechanism involved intracellular enzymes cytochrome P450

monooxygenase and epoxide hydrolase to yield trans-dihydrodiols, phenols, quinones, and

dihydrodiol-epoxides (Cerniglia & Sutherland, 2001; Cerniglia, 1997). Miller and Ramos

(2001) also reported that benzo[a]pyrene can be oxidized by cytochrome P450

monooxygenases in the mammalian liver.