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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.
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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
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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
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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
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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
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,. 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
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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
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XIV
XV11
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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
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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
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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
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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
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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
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