LIGNINOLYTIC ENZYMES ACTIVITIES DURING BIODEGRADATION OF OIL PALM EMPTY FRUIT BUNCH (EFB) BY

LOCALLY ISOLATED WHITE ROT FUNGI

Lee Wak Ha

Master of Science 2013

Pusat Khidmat MakJumat Akademik UNIVERSITJ MALAYSIA SARAWAK

LIGNINOLYTIC ENZYMES ACTIVITIES DURING BIODEGRADATION OF OIL PALM EMPTY FRUIT BUNCH (EFB) BY LOCALLY ISOLATED WHITE ROT

FUNGI

LEEWAKHA

A thesis submitted in fulfillment of the requirements for the degree of

Master of Science

Faculty of Resource Science and Technology UNIVERSITY MALAYSIA SARA W AK

2013

DECLARATION

I hereby declare that no portion of this thesis has been submitted in support of an application for

another degree or qualification of this or any other university or institution of higher learning.

_.W ·H

(Lee Wak Ha)

Date:

ACKNOWLEGEMENT

First at all, I would like to thank God for all the blessings and strength that He had granted me

upon completing this project. Secondly, I would like to express my appreciations to my

supervisors, Dr. Awang Ahmad Sallehin bin Awang Husaini and Dr. Mohd Hasnain Md Hussain,

for their support, advice and help in the completion of this project. I also take this opportunity to

thank them for spending their time in helping me to prepare this manuscript.

I would like to express my sincere gratitude to Ms. Rohanie Bohan and Ms. Angelyne

Malaya for their kind assistance, helpful advices, and supports in completing this project. My

appreciation is extent to laboratory assistants for their technical help and friendship they offered

while conducting the research and during the study. My sincere thanks also go to all my lab

mates for their assistance and encouragement in the times of difficulties.

I am indebted to my family for their love, care and providing me with the opportunity to

further my study.

Last but not least, I am deeply grateful to all my fellow friends in the faculty for their

helpful initial study, priceless suggestion, their companionship and the good times we had

together.

ii

ABSTRACT

Malaysia is one of world's largest palm oil producing countries and producing more than

eighteen tonnes of crude palm oil. One of the significant problems in the palm fruit processing is

managing of the wastes generated during the processes. On average, for every ton of fresh fruit

bunches will produced around two hundred kg of empty fruit bunched (EFB). The abundance of

EFB has created an important environmental issue such as fouling, attracting of pests and carbon

dioxide and methane emissions. In order to minimize the abundant disposal of this waste and

environmental problems, new applications on the use of abundant biomass as urgently required.

The experimental work of this study aims to provide knowledge and infonnation on the profiling

and enzyme characterization of fungi as candidate for lignin biodegradation during

biodegradation of oil palm empty fruit bunch by white rot fungi. In this research, fifty five fungal

isolates were successfully isolated and only ten isolates were successfully decolourised two dyes,

Remazol Brilliant Blue R (RBBR) and Orange II. Cerrena sp., Athelia pellicularis (H13W) and

Basidiomycetes sp. HKB30 (F9W), which gave the highest yield, were selected for further

analysis in liquid medium and lignin and Mn2+-oxidizing peroxidases and laccase, activities were

assayed. Lignin biodegradation trials were perfonned on the natural substrate, oil palm empty

fruit bunch. The profiles and patterns of the peroxidase enzymes secreted during the lignin

biodegradation process were studied. Results revealed that all three fungal isolates secretes

ligninolytic enzymes during their four weeks growth period. Cerrena sp. has the highest activities

towards lignin peroxidase on crush EFB recorded 9.892 U mL-1, while Basidiomycete sp. HKB30

(F9W) is active in secreting lignin peroxidase on uncrush EFB recorded 7.627 U mL- 1• As for

iii

manganese peroxidase activity, Cerrena sp. has the highest activities in the crush and uncrushed

EFB, recorded 4.032 U mL-1 and 3.882 U mL-1, respectively. Overall, all these three fungal

isolates exhibit the lowest enzymatic activities towards laccase. Lignin biodegradation was

carried out for a period of four weeks to examine the activities of fungi in performing the

degradation of lignin in EFB at one week intervals. The original lignin content in crush EFB is

28.06% were reduced by fungal pretreatments to values of 12.27%, 4.01% and 10.85% after

biotreated with Cerrena sp., Basidiomycete sp. HKB30 (F9W) and Athelia pellicularis (H13W),

respectively. As for the uncrushed EFB, the origina11ignin content is 35.36%. The values were

reduced to 16.97%, 22.19% and 19.02% after biotreated with Cerrena sp., Basidiomycete sp.

HKB30 (F9W) and Athelia pellicularis (H13W), respectively. The structure ofEFB (longitudinal

section) before and after biotreated by the fungi was viewed under Scanning Electron Microscope

(SEM) and it showed that the surface structure for both untreated crush and un crushed EFB

consisted of firmly bound threads with smooth surface along the structure. Besides, most of the

outer surface of biotreated crush and uncrush EFB seems has been altered with the presence of

many holes. Further studies needs to be carried out in. their genetic manipulation studies in

improving and exploiting them as biodegradation agent. Moreover, further investigations are

needed to examine the lignin degradative enzymes activities particularly the oxidative enzymes

that are involved especially on the characteristic, enzymology and molecular biology of the

lignino1ytic system employed by the indigenous fungi. Furtliermore, the role and properties of the

ligninolytic enzymes should be clarified in more detail to examine their significance for the

composting processes.

Key words: lignin peroxidase, manganese peroxidase, 1accase, 1ignino1ytic

iv

Aktiviti-aktiviti enzim ligninolitik semasa biodegradasi tan dan kosong buah kelapa sawit (EFB) oleh kulat pereput putih tempatan

ABSTRAK

Malaysia adalah salah satu negara yang terbesar dalam pemprosesan minyak sawit di dunia dan

menghasilkan lebik kurang lapan belas tan minyak sawit. Salah satu masalah yang penting

dalam pemprosesan buah kelapa sawit ialah pengurllsan sisa-sisa yang dihasilkan semasa

pemprosesan hlang. Secara purata, pemperosesan satu ton metric buah tandan kelapa sawit

akan menghasilkan kira-hra 200kg tandan kosong buah kelapa sawit (EFB). Tandan kosong

buah kelapa sawit ini telah menwujud satl{ isu alam sekitar yang penting seperti menarik perosak

dan pelepasan karbon dioksida dna metana. Dalam usaha mengurangkan pelupusan sisa yang

banyak ini dan masalah alam sehtar, aplikasi baru mengenai penggllnaan biojisim ini adalah

amat diperlukan. Jadi, kajian eksperimental ini bertujuan untuk memberi pengetahuan dan

maklumat mengenai profil and ciri-ciri enzim kulat sebagai calon untuk biodegradasi lignin

semasa biodegradasi tandan kosong buah kelapa sawit oleh kulat reput putih. Dalam kajian ini,

lima puluh lima kulat telah berjaya diperolehi dan hanya sepuluh pencilan yang berjaya

menyahwamakan kedua-dua pewama ini, Remazol Brilliant Blue R (RBBR) dan Orange II.

Cerrena sp., Athelia pellicularis (H13W) dan Basidiomycetes sp. HKB30 (F9W) telah

memberikan hasil tertinggi, telah dipilih untuk analisa lanjut penghasilan enzim di dalam media

cecair bagi enzim lignin peroksidase, mangan peroksidase dan lakase. Ujikaji biodegradasi

lignin telah dilakukan ke atas substrat semulajadi, menggunakan tandan kosong buah kelapa

sawit. Profil dan corak perembesan enzim peroksidase semasa proses biodegradasi lignin telah

dikaji. Hasil kajian menunjukkan bahawa ketiga-tiga kulat pendlan merembeskan enzim

ligninolitik sepanjang empat minggu tempoh pertumbuhan. Cerrena sp. telah menghasilkan

v

aktiviti lignin peroksidase tertinggi pada substrat EFB yang dihancurkan iaitu 9.892 U mL-J,

sementara Basidiomycete sp. HKB30 (F9W) aktif dalam merembeskan lignin peroksidase pada

EFB yang tidak dihancurkan iaitu, 7.627 U mr-J. Bagi aktiviti enzim mangan peroksidase,

Cerrena sp. merekodkan aktiviti tertinggi dalam EFB yang dihancukan dan tidak dihancurkan,

liaitu 4.032 U mr- dan 3.882 U mL-J. Secara keseluruhannya, ketiga-tiga kulat pencilan

menunjukkan al.:tiviti enzim terendah pada enzim lakase. Ujikaji biodegradasi lignin telah

dijalankan untuk tempoh selama empat minggu bagi memeriksa aktiviti-aktiviti kulat dalam

biodegradasi lignin pada substrat EFB dengan persampelan dijalankan pada selang satu minggu.

Kandungan lignin asal dalam substrat EFB yang dihancurkan adalah 28.06% telah diturunkan

oleh kulat kepada 12.27%, 4.01% dan 10.85% selepas didegradasikan dengan Cerrena sp.,

Basidiomycetes sp. HKB30 (F9W) dan Athelia pellicularis (H13W). Bagi substrat EFB yang

tidak dihancurkan, kandungan asal lignin adalah 35.36%. jumlah kandungan lignin telah

diturunkan kepada 16.97%, 22.19% dan 19.02% selepas didegradasikan dengan Cerrena sp.,

Basidiomycetes sp. HKB30 (F9W) dan Athelia pellicularis (H13W). Stn/ktur EFB (keratan

memanjang) sebelum dan selepas proses bioderadasi oleh kulat telah dikaji menggunakan

mikroskop pengimbasan electron (SEM) didapati bahawa struktur permukaan untuk kedua-dua

substrat EFB yang dihancurkan dan tidak dihancurkan yang belum dirawat mengandul1gi

benang yang kukuh terikat pada permukaan licin di sepanjang struktur. Ujikaji yang sarna ke

atas substrat EFB yang dihancurkan dan tidak dihartcurkan selepas proses biodegradasi

mendapati kebanyakan daripada permukaan luar substrat tersebut kelihatan telah diubahsuai

dengan kehadiran lubang yang banyak. Kajian lanjutan perlu dijalankan dalam manipulasi

genetik kulat dari segi memperbaiki dan mengeksploitasi kulat sebagai ejen biodegradasi. Selain

itu, kajian bagi aktiviti enzim degradasi lignin juga diperlukan terutamanya enzim oksidatiJ yang

vi

,. I

terlibat dari segi ciri-ciri, enzimologi and sistem molekul biologi /igninolitik yang digunakan

oleh kulat. Tambahan pula, peranan dan sifat-sifat enzim, ligninolitik perlu dijelaskan dengan

lebih terperinci untuk mengkaji kepentingan mereka untuk proses pengkomposan.

Kata kunci: lignin perosidase, mangan perosidase, lakase, ligninolitik

Vll

Pusat Khidmat Maklumat Akademik UNlVERSm MALAYSIA SAKAWAK

TABLE OF CONTENTS

DECLARATION

ACKNOWLEGMENT

ABSTRACT

ABSTRAK

TABLE OF CONTENT

LIST OF FIGURES

LIST OF TABLES

LIST OF ABBREVIATIONS

CHAPTER 1 INTRODUCTION

CHAPTER 2 LITERATURE REVIEW

2.1 Biodegradation

Page

11

III

v

viii

XIV

XV11

xviii

6

6

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2.2 Fungi 7

2.2.1 Basidiomycetes 9

2.2.1.2 White rot fungi 10

2.3 Heterokaryosis 11

2.4 Solid substrate fermentation (SSF) 11

2.4.1 Inoculation of the substrate 12

2.4.2 Effect of particle size l3

2.5 Lignin l3

2.5.1 Structure of lignin 15

2.5.2 Microbial degradation of lignin 16

2.5.3 Fungalligninolytic mechanisms 18

2.5.3 .1 Lignin peroxidases 19

2.5 .3.2 Manganese peroxidases 20

2.5.3.3 Laccase 21

2.6 Determination of lignin content 22

2.7 Empty fruit bunch (EFB) as lignocellulosis substrate 23

2.8 Composting of empty fruit bunch (EFB) 23

2.9 Biodegradation of empty fruit bunch (EFB) by using fungi 25

CHAPTER 3 MATERIALS AND METHODS 26

3.1 Isolation and screening of white rot fungi for potential lignin 26

degradation

ix

3.1.1 Collecting of samples 26

3.1.2 Preparation of culture media 26

3.1.3 Isolation of fungi 27

3.1.4 Culture conditions 27

3.1.5 Primary screening method on solid medium 28

3.1.6 Quantitative analysis in liquid media 28

3.1.7 Ligninolytic enzyme activities 29

3.1.7.1 Lignin peroxidase (LiP) activity 29

3.1.7.2 Manganese peroxidase (MnP) activity 30

3.1.7.3 Laccase activity 30

3.1.8 Data analysis 31

3.2 Fungal strains identification 32

3.2.1 Morphological characterization 32

3.2.2 Molecular identification of fungal isolates 32

3.2.2.1 Fungal DNA extraction 32

3.2.2.2 Agarose gel electrophoresis 33

3.2.2.3 Polymerase chain reaction (PCR) using ITS 33

universal primer

3.2.2.4 Purification ofPCR ptoduct 34

3.3 Lab scale lignin biodegradation of oil palm empty fruit bunch 34

(EFB)

3.3.1 Microorganism 34

x

I ,.

3.3.2 Culture condition 34

3.3 .3 Lignin biodegradation 35

3.3.4 Enzymes extraction from biotreated oil palm empty fruit 36

bunch (EFB)

3.3.4.1 Ligninolytic enzyme activities 36

3.3.4.1.1 Lignin peroxidase (LiP) activity 36

3.3.4.1.2 Manganese peroxidase (MnP) 37

activity

3.3.4.1.3 Laccase activity 37

3.3.5 Determination of oil palm empty fruit bunch (EFB) 37

weight loss

3.3.5.1 Determination of lignin weight loss 38

3.3 .6 Scanning electron microscope (SEM) analysis 38

CHAPTER 4 RESULTS AND DISCUSSION 39

4.1 Isolation and screening of white rot fungi for potential lignin 39

degradation

4.1.1 Isolation of fungi 39

4.1.2 Screening for ligninolytic white rbt fungi 39

4.1.3 Ligninolytic enzymes activities 44

4.1.4 ANOV A analysis of enzymes activities 47

4.2 Morphological identification of selected fungal isolates 47

Xl

4.3 Molecular identification of the selected fungal isolates 51

4.3.1 DNA isolation 51

4.3.2 peR amplification of the Internal Transcribed Spacer 50

(ITS) Region

4.3.3 Molecular identification of the fungal isolates 53

4.4 Lab scale lignin biodegradation of oil palm empty fruit bunch 54

(EFB)

4.4.1 Microorganism and culture condition 54

4.4.2 Morphological characteristics of biodegraded EFB 54

4.4.3 Enzymes extraction from biotreated oil palm empty fruit 56

bunch (EFB)

4.4.3.l Ligninolytic enzyme activities 56

4.4.3.1.1 Lignin peroxidase (LiP) activity 57

4.4.3 .1.2 Manganese peroxidase (MnP) 60

activity

4.4.3.1.3 Laccase (Lac) activity 62

4.4.4 Determination of oil palm empty fruit bunch (EFB) lignin 66

weight loss

4.4.5 Scanning Electron Microscope (SEM) Analysis 70

CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS 76

xii

79 REFERENCES

APPENDIX A

APPENDIXB

xiii

LIST OF FIGURES

PAGE Figure 1 Structure of a wood cell. (S3 = secondary wall 3; S2 = secondary 15

wall 2; S 1 = secondary wall 1; P = primary wall; ML = middle lamella)

Figure 2 A phenylpropanoid unit and precursors of lignin. From left to right: 16 p-coumaryl alcohol, coniferyl alcohol, sinapyJ alcohol, and a model for the numeration of the carbon skeleton (Helsinki, 2002).

Figure 3 Ten days old fungal liquid culture was used as inoculums to 35 inoculate the oil palm empty fruit bunch (EFB).

Figure 4 Sterile oil palm EFB in 250 ml Erlenmeyer flask (A - crushed 36 EFB; B- uncrush EFB).

FigureS F9W on Day 3 of incubations on agar plates. (plate A: MMS agar 41 plate (control); plate B: RBBR; plate C: Orange II).

Figure 6 Cerrena sp. on Day 3 of incubations on agar plates. (plate A: MMS 42 agar plate (control); plate B: RBBR; plate C: Orange II).

Figure 7 H13W on Day 3 of incubations on agar plates. (plate A: MMS agar 42 plate (control); plate B: RBBR; plate C: Orange II).

Figure 8 Peroxidases enzyme activities (U mL- 1) in minimal mineral salt 45

media for 14 days of incubation period in batch culture (p-values > 0.05)

Figure 9 H13W on MEA. (A) Reverse colony surface. (B) Colony surface. 48

Figure 10 F9W on MEA. (A) Reverse colony surface. (B) Colony surface 48

Figure 11 Fungi - H13W 49

Figure 12 Fungi test strain - H13W: (a) basal hyphae (~) part ofhymenium 49 and subhymenial hyphae (c) basidium and spores

Figure 13 Fungi test strain - F9W: (a) Tramal generative hyphae (b) basidio 50 and basidiospores

Figure 14 Formation of clamp cell: (A) Dikaryotic hypha, arrow shows 51 direction of hyphal tip growth. (B) Clamp cell growing backward, nuclei undergoing synchronous division. (C) Mature clamp (Eric, e

XIV

at., 2004)

Figure 15 The genomic DNA bands of 2 selected fungal test strains when visualized in 1 % (w/v) agarose gel. Lane 1 ­ H13W, Lane 2­F9W.

52

Figure 16 peR product generated by the ITS 4 and ITS 5 universal primers for the selected fungal test strains when visualized using 1.5% (w/v) agarose gel. M - 100 bp molecular weight marker (DNA ladder), Lane 1 ­ F9W, Lane 2 ­ H13W, N - negative control.

53

Figure 17 The EFB completely covered by mycelia map. 55

Figure 18 The differences between biotreated EFB (A) and untreated EFB (B).

56

Figure 19 Lignin peroxidase activity on crush EFB over 4 weeks incubation period in batch cultures. There was no significant (p-values >0.05) difference between the fungal isolates for their LiP activitiy on crush EFB in minimal mineral salt medium. Each data is represented by the mean of three replicates

58

Figure 20 Lignin peroxidase activity on uncrush EFB over 4 weeks of incubation period in batch cultures. There was no significant (p­values> 0.05) difference between the mean values. Each data is represented by the mean of three replicates.

58

Figure 21 Manganese peroxidase activity on crush EFB over 4 weeks of incubation period in batch cultures. There was significant (p-values > 0.05) difference between the fungal isolates for their MnP activitiy on crush EFB in minimal mineral salt medium. Each data is represented by the mean of three replicates.

61

Figure 22 Manganese peroxidase activity on uncrush EFB over 4 weeks of incubation period in batch cultures. There was significant (p-values > 0.05) difference between the mean values. Each data is represented by the mean of three replicates ..

62

Figure 23 Laccase activity on crush EFB over 4 weeks of incubation period in batch cultures. There was significant (p-values < 0.05) difference between the fungal isolates for their Lac enzyme activity on crush EFB in minimal mineral salt medium. Each data is represented by the mean of three replicates.

63

Figure 24 Laccase activity on uncrush EFB over 4 weeks of incubation 64

xv

period in batch cultures. There is no significant (p-values > 0.05) difference between the mean values. Each data is represented by the mean of three replicates.

Figure 25 Lignin content left of crush EFB biotreatment by Cerrena sp., Basidiomycetes sp. F9W and Athelia pellicularis H13W over degradation periods ranging from 1 to 4 weeks.

67

Figure 26 Lignin content left ofuncrush EFB biotreatment by Cerrena sp., F9W and H13W over degradation periods varying from 1 to 4 weeks.

68

Figure 27 SEM on untreated uncrush EFB as control. 71

Figure 28 SEM on untreated crush EFB as control. 71

Figure 29 SEM of Basidiomycete sp. F9W biodegradation pattern on uncrush EFB at 4 weeks of biodegradation.

71

Figure 30 SEM of Basidiomycete sp.F9W biodegradation pattern on crush EFB after 4 weeks of biodegradation.

72

Figure 31 SEM ofAthelia pellicularis H13W biodegradation pattern on uncrush after 4 weeks of biodegradation.

72

Figure 32 SEM ofAthelia pellicularis H13W biodegradation pattern on crush EFB after 4 weeks of biodegradation.

73

Figure 33 SEM of Cerrena sp. biodegradation pattern on uncrush EFB after 4 weeks ofbiodegradation.

73

Figure 34 SEM of Cerrena sp. biodegradation pattern on crush EFB after 4 weeks of biodegradation.

74

xvi

LIST OF TABLE

Table 1 Duration for complete Remazol Brilliant Blue R (RBBR) and Orange II dyes decolourization by white rot fungi for 14 days at 27°C.

PAGE 40

Table 2 Fungal test isolates and the species types identified through BLAST programme in NCBr GenBank.

54

XVIl

LIST OF ABBREVIATIONS

Bp Base Pairs

em Centrimeter

CO2 Carbon dioxide

DNA Deoxyribonucleic Acid

DMP 2,6-dimethoxyphenol

dNTP Deoxynucleotide triphosphate

EFB Empty fruit bunches

g Gram

H2SO4 Sulphuric acid

ITS Internal Transcribed Spacer

LiP Lignin Peroxidase

M Molar

ME Malt Extract

MEA Malt Extract Agar

Min Minute

mM MiliMolar

MMS Minimum Mineral Salt

MnP Manganese Peroxidases

00 Optical Density

PCR Polymerase chain reactions

rpm Revolution per minute

SSF solid substrate fermentation

VA Veratryl Alcohol

UV Ultraviolet

°c Degree Celsius

xviii

CHAPTER 1

INTRODUCTION

Oil pabn (Elaeis guineensis) originates from West Africa where it grows in the wild and

is presently developed into an agricultural crop. It was introduced to Malaysia by the British in

early 1870's as an ornamental plant (Malaysia Pabn Oil Council, 2012). In 1917, the first

commercial oil pabn estate was set up in Tennamaran Estate in Selangor, and this laid the

foundations for the vast oil pabn plantations and the oil pabn industry in Malaysia (Malaysia

Palm Oil Council, 2012). In 2011, the oil pabn planted area reached 5 million hectares and are

producing 18.91 million tonnes of crude pabn oil and 2.14 tonnes of crude palm kernel oil

(Anonymous, 2012).

Oil palm is a major source of edible oil which is extracted from its fruits . Every year the

oil palm plantation yields a staggering amount of harvestable biomass (some 50 to 70 tonnes per

hectare per year), and only 10 percent of this total results in the finished products such as pabn oil

and palm kernel oil. Until recently, the remaining 90 percent (empty fruit bunches, fibers, fronds,

trunks, kernels, pabn oil mill effluent) was discarded as waste, either burned in the open air or

left to settle in waste ponds (Salathong, 2007).

Malaysia alone can generate large amounts of oil pabn biomass from the palm oil industry,

for example 500 million tonnes (green) of felled trunk in 2000, 36 million tonnes per year of

fronds from pruning and replanting (Wan Zahari et al., 2004) and 19.03 million tonnes (wet

weight basis) of empty fruit bunches (EFB) in 2007 (Astimar et ai., 2009). As a result, the palm

oil processing industry's waste contributes significantly to carbon dioxide and methane emissions.

In order to minimize the abundant disposal of this waste and environmental problems, new

applications on the use of abundant biomass are urgently required.

Oil palm empty fruit bunch (EFB) is the by-product generated from palm oil mills, and

after the oil is extracted. EFB contains polymeric lignocellulosic components such as cellulose,

hemicelluloses and lignin. According to Abdul Aziz et ai (1989), the EFB consists of 60 percent

of cellulose and hemicelluloses. Among these components, lignin is probably the most

recalcitrant to biodegradation (Hammel, 1997). This is due to the link of lignin with cellulose and

hemicelluloses, forming a physical seal that is impenetrable towards enzymes and

microorganisms (Howard et aI., 2003). In other words, it is also consistent with its biological

functions, whereby lignin gives vascular plants the rigidity they need to stand upright and to

protect their polysaccharides (cellulose and hemicelluloses) from attack by other organisms.

Lignin is the most abundant aromatic compound on earth, comprising of 15 percent to 30

percent of woody plant cell walls, and is second only to cellulose in its contribution to living

terrestrial biomass (Crawford, 1981). When vascular plants die or drop litter, lignified organic

carbon is incorporated into the top layer of the soil. This recalcitrant material has to be broken

down and recycled by microorganisms to maintain the earth ' s carbon cycle and carbon would

eventually be irreversibly sequestered as lignocelluloses (Hammel, 1997). Undegraded

ligninocellulose, for example in the form of straw, has a deleterious effect on soil fertility because

2

decomposing (as opposed to already decomposed) lignocelluloses supports high populations of

microorganisms that may produce phytotoxic metabolites. High microbial populations in

undecomposed litter also compete with crop plants for soil nitrogen and other nutrients (Lynch

and Harper, 1985). Consequently, the network oflignin must be decomposed in order to allow for

enzymatic conversion of lignocellulosic materials to fermentable sugars (Sun and Cheng, 2002).

The aromatic polymer lignin is a highly branched, and its heterogeneous three­

dimensional structure is made up of phenylpropanoid units which are interlinked through a great

variety of different bonds (Brunow, 2001). According to Kirk and Farrell (1987), fungi are

recognized for their superior aptitudes to produce a large variety of extracellular enzymes. The

organisms which principally responsible for lignocelluloses degradation are aerobic filamentous

fungi, and the most rapid degraders in this group are basidiomycetes.

Basidiomycetes, white rot fungi are the only organism capable of mineralizing lignin

efficiently. These fungi produce various combinations of non-specific and oxidative extracellular

enzymes which are directly involved in initiating the depolymerization of lignin which are lignin

peroxidases (LiP), manganese peroxidases (MnP) and laccase (Lac). Some white rot fungi

produce all these enzymes, while others produce only one or two of them (Boer et al., 2004) .

According to Have et al. (1998), these peroxidases and' hydrogen peroxide (H202) generate

enzymes that work together to initiate lignin oxidation, with the most potent peroxidases having

the capability to oxidize lignin with a high ionization potential which is the lignin peroxidases.

3

In previous research, white rot fungi and their enzymes were being studied extensively for

their application in the degradation of aromatic pollutants which caused environmental problems

like pulp and paper mills (Machii et al., 2004), olive mill wastewater (Jaouani et al., 2003),

polycyclic aromatic hydrocarbons (PAHs) (Clemente et al., 2001), chlorinated phenols,

polychlorinated biphenyls (Sato et al., 2002), dioxins, pesticides, explosives and dyes

(Wesenberg et al., 2003, Levin et al.,2003, and Levin et al. ,2004). Frequently, more than one

isoform of ligninolytic enzymes are expressed by different taxa and culture conditions. These

features are important in the process design and optimization of fungal treatment of effluents.

Purified Lac, LiP and MnP are potential enzymes for various industrial applications (Dhouib et

al., 2005).

Majority of the previous studies focused mainly on the lignin degrading enzymes of

Phanerochaete chrysosporium, which is the model species most intensively studied (Krejci, et al.,

1991). However, the possible practical applications of the model fungus does not always allow

for the optimum culture conditions to be fulfilled. Therefore, there is a growing interest in

studying the lignin-modifying enzymes of a wide array of white rot fungi, not only from the

standpoint ofcomparative biology but also with the expectation of fmding better lignin degrading

systems for use in various biotechnological applications.

Tropical white rot fungi strains, which are widely represented in the forest of Sarawak,

are the least studied with respect to their biodegradative capabilities. These tropical

basidiomycetes are tolerant to harsh tropical environmental conditions (Tekere et al. , 2001), thus

4