Faculty of Resource Science and Technology SCREENING AND CHARACTERIZATION OF LIGNINOLYTIC FUNGUS FROM MIXED ORGANIC MATTER

Faculty of Resource Science and Technology SCREENING AND CHARACTERIZATION OF LIGNINOLYTIC FUNGUS FROM MIXED ORGANIC MATTER Lobo Ak Rodab Bachelor o...
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Faculty of Resource Science and Technology

SCREENING AND CHARACTERIZATION OF LIGNINOLYTIC FUNGUS FROM MIXED ORGANIC MATTER

Lobo Ak Rodab

Bachelor of Science with Honours (Resource Biotechnology) 2007

SCREENING AND CHARACTERIZATION OF LIGNINOLYTIC FUNGUS FROM MIXED ORGANIC MATTER

Faculty of Resource Science and Technology LOBO AK RODAB

This project is submitted in partial fulfillment of the requirements for the degree of Bachelor of Science with Honours (Resource Biotechnology)

Resource Biotechnology Programme Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK 2007

ACKNOWLEDGEMENT

First of all, I would like to thank God for His blessings and great opportunities He gave me upon the completion of this project. My sincere appreciation to my supervisor Dr. Awang Ahmad Sallehin bin Awang Husaini for his time, advisory, guidance and his continuous support throughout this project. Besides that, I would like to thank Assoc. Prof. Dr. Sepiah Muid, Dr. Hairul Azman Roslan, Dr. Edmund Sim, and Assoc. Prof. Dr. Kasing Apun for important information and permission to use some of the facilities in their laboratories.

I am grateful to Mr. Jaya Seelan Sathiya Seelan, Mr. Ang Chung Huap, Miss Pearl, Miss Julie and all master students in FSTS faculty for their useful experiences and knowledge, helps, advices and support inside the lab. My sincere appreciation to the management of the faculty and university for providing the facilities and comfortable working environment, especially Mr. Azis for providing me with the palm oil sample, and also to Madam Sheila for preparing equipments and materials in the Molecular lab. My gratefulness also goes to all my supportive friends and course mates for their companionship and great times together.

My special thanks to Dato Hajjah Raziah Mahmud-Geneid for her sponsorship, kindness and support throughout my studies in UNIMAS. Last but not least, I am deeply touched by the support and sacrifice shown by my beloved family.

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TABLE OF CONTENT Page ACKNOWLEDGEMENT

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TABLE OF CONTENT

ii

LIST OF FIGURES

v

LIST OF TABLES

vi

LIST OF ABBREVIATIONS

vii

ABSTRACT

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CHAPTER 1 INTRODUCTION

1

CHAPTER 2 LITERATURE REVIEW 2.1

Lignin

4

2.2

Ligninolytic fungi

4

2.3

Enzymes secreted by ligninolytic fungi

6

2.3.1

Lignin peroxidase (LiP)

6

2.3.2

Manganese peroxidase (MnP)

7

2.3.3

Laccase (Lcc)

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2.3.4

Cellulase (Cell)

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2.4

Morphological identification of fungal isolates

2.5

Molecular identification of fungal isolates by ITS primer

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CHAPTER 3 MATERIALS AND METHODS 3.1

Materials

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3.2

Methods

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3.2.1

Collection of agriculture’s waste samples

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3.2.2

Fungal isolation

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3.2.3

Preliminary screening of ligninolytic fungi by dye decolorization test

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3.2.4

Morphology identification of fungal isolated

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3.2.5

Enzyme biochemical test in minimal media

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3.2.6

3.2.7

Molecular identification of selected fungal isolates 3.2.6.1 Fungal DNA extraction

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3.2.6.2 Electrophoresis analysis

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3.2.6.3 DNA purification

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3.2.6.4 DNA quantification

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3.2.6.5 PCR amplification using ITS Universal Primer

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3.2.6.6 Purification of PCR products

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3.2.6.7 PCR product sequencing

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Incubation period study on lignin degradation for selected fungal isolates

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3.2.7.1 Determination of crude ligninolytic enzymes activity

3.2.8

3.2.7.1.1 Lignin peroxidase (LiP)

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3.2.7.1.2 Laccase (Lcc)

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3.2.7.1.3 Manganese peroxidase (MnP)

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3.2.7.1.4 Cellulase (Cell)

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3.2.7.2 Data analysis

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SDS-PAGE analysis

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CHAPTER 4 RESULTS 4.1

Fungal isolation

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4.2

Preliminary screening of ligninolytic fungi by dye decolorization test

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4.3

Morphology identification of fungal isolates

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4.4

Biochemical test for ligninolytic enzyme in minimal media

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4.5

Molecular identification of selected fungal isolates 4.5.1

Fungal DNA extraction

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4.5.2

DNA quantification

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4.5.3

PCR amplification using ITS Universal Primer

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4.5.4

PCR product sequencing

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4.6

Incubation period study on lignin degradation for selected fungal isolates 4.6.1

4.7

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Determination of crude ligninolytic enzymes activity 4.6.1.1 Lignin peroxidase (LiP)

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4.6.1.2 Laccase (Lcc)

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4.6.1.3 Manganese peroxidase (MnP)

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4.6.1.4 Cellulase (Cell)

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SDS-PAGE analysis

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CHAPTER 5 DISCUSSIONS 5.1

Preliminary screening of ligninolytic fungi by dye decolorization test

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5.2

Biochemical test for ligninolytic enzyme in minimal media

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5.3

Molecular identification of selected fungal isolates

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5.4

Incubation period study on lignin degradation for selected

5.5

fungal isolates

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SDS-PAGE analysis

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CHAPTER 6 CONCLUSIONS & RECOMMENDATION

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REFERENCES

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APPENDICES  APPENDIX A  APPENDIX B  APPENDIX C  APPENDIX D  APPENDIX E

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LIST OF FIGURES Page Figure 1: Dye decolorization test for five difference isolates

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Figure 2: Morphology and characteristics of Cladosporium species

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Figure 3: Morphology and characteristics of Aspergillus sydowii

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Figure 4: Morphology and characteristics of Fusarium solani

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Figure 5: Morphology and characteristics of Pestalotiopsis species

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Figure 6: Distribution of four difference enzyme activities for each selected fungal isolates

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Figure 7: Agarose gel electrophoresis result for DNA extraction of three fungal isolates

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Figure 8: Agarose gel electrophoresis of amplified PCR products

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Figure 9: Conical flasks containing media, sawdust and homogenized mycelial plugs and crude enzyme extracts obtained after the incubation period

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Figure 10: LiP activity over days of incubation

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Figure 11: Laccase activity over days of incubation

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Figure 12: MnP over days of incubation

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Figure 13: Cellulase activity over days of incubation

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Figure 14: Protein analysis using SDS-PAGE

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Figure 15: Glucose standard curve

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LIST OF TABLES Page Table 1: PCR composition for ITS 4 and ITS 5.

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Table 2: Parameters for PCR amplification.

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Table 3: Diameter sizes of clear halo zones formed on agar plates inoculated with five difference fungal isolates.

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Table 4: The concentration and purity of the DNA yields for the 3 fungal isolates

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Table 5: List of fungal isolates from three difference agriculture’s wastes from three difference locations.

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Table 6: Lists of selected fungal isolates for screening and microscopic works

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Table 7: Biochemical test for LiP, MnP, laccase and cellulase using minimal media.

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Table 8: LiP, MnP, laccase and cellulase activities in U/mL.

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Table 9: Absorbance reading for glucose at A540

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Table 10: Lignin peroxidase (LiP) assay according to days of incubation

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Table 11: Laccase (Lcc) assay according to days of incubation

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Table 12: Manganese peroxidase (LiP) assay according to days of incubation

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Table13: Cellulase (Cell) assay according to days of incubation

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Table 14: LiP activities (U/mL) and ANOVA analysis for three fungal isolates

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Table 15: Lcc activities (U/mL) and ANOVA analysis for three fungal isolates

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Table 16: MnP activities (U/mL) and ANOVA analysis for three fungal isolates

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Table 17: Cell activities (U/mL) and ANOVA analysis for three fungal isolates

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LIST OF ABBREVIATIONS

BLAST

Basic Local Alignment Search Tool

bp

Base pairs

cm

Centimeter

Da

Dalton

DNA

Deoxyribonucleic acid

g

Gram

H2O

Water

ITS

Internal Transcribed Spacer

kb

Kilobase pairs

M

Molar or molarity (moules of solute per liter of solution)

ml

Mililiter

mM

Milimolar

NCBI

National Centre for Biotechnology Information

μg

Microgram

μl

Microliter

PCR

Polymerase Chain Reaction

Taq Polymerase

Thermus aquaticus DNA polymerase

TAE

Tris-EDTA buffer

V

Volts

%

Percent

°C

Degree Celsius

Zygo

Zygomycetes

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Screening and characterization of ligninolytic fungus from mixed organic matter Lobo Ak Rodab Resource Biotechnology Programme Faculty of Resource Science and Technology University Malaysia Sarawak 2007

ABSTRACT Three difference types of mixed organic matter from agriculture's wastes have led to successful isolation of 24 indigenous fungal isolates. Among these isolates, Aspergillus sydowii strain NRRL 250 (OP11), Pestalotiopsis theae isolate P145 (PA6) and Cladosporium cladosporioides strain Hu01 (PA21) were further selected for lignin degradation analysis, where all three isolates have consistent ligninolytic enzymes activities for all four enzymes tested earlier in minimal media. PA21 also show active Congo Red dye degradation activity as compared to other four difference species all positive for the test. PA21 was the best Lignin peroxidase (LiP) and laccase producing fungi. Cellulase is the highest enzyme secreted for all fungal isolates. Enzyme activities ranged from 44.16-337.87 U/mL (LiP), 1.55-50.03 U/mL (laccase), 23.43-276.64 U/mL (MnP), and 184.0-1449.0 U/mL (cellulase). Highest enzyme activities for LiP and cellulase achieved by isolate OP11 on day 11, while for Manganese peroxidase (MnP) achieved by isolate PA6 on day 5 and for laccase by isolate PA21 on day 5. Keywords: Mixed organic matter, indigenous fungi, lignin degradation, ligninolytic enzymes, dye degradation

ABSTRAK Terdapat 24 jenis kulat ditemui daripada tiga jenis bahan organik campuran yang diperolehi dari kawasan pertanian. Daripada semua jenis kulat ini, Aspergillus sydowii strain NRRL 250 (OP11), Pestalotiopsis theae isolate P145 (PA6) dan Cladosporium cladosporioides strain Hu01 (PA21) telah dipilih untuk analisa degradasi lignin berdasarkan aktiviti-aktiviti enzim ligninolitik yang seragam di dalam medium minima. PA21 juga menunjukkan aktiviti degradasi ke atas pewarna Congo Red lebih baik daripada empat lagi jenis kulat yang juga positif ke atas ujian tersebut. PA21 ialah penghasil enzim Lignin peroksida (LiP) dan laccase yang terbaik. Cellulase ialah enzim yang paling banyak dihasilkan oleh ketiga-tiga jenis kulat tersebut Aktiviti-aktiviti enzim adalah dalam kadar 44.16337.87 U/mL (LiP), 1.55-50.03 U/mL (laccase), 23.43-276.64 U/mL (MnP), and 184.0-1.449.0 U/mL (cellulase). Aktiviti-aktiviti enzim bagi LiP dan cellulase paling tinggi dicatatkan oleh OP11 pada hari yang ke-11, manakala bagi Mangan peroksida (Mn)P paling tinggi dicatatkan oleh PA6 pada hari ke-5 dan bagi laccase paling tinggi pada hari ke-5 oleh PA21. Kata kunci: Bahan organic campuran, kulat, degradasi lignin, enzim ligninolitik, degradasi pewarna

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CHAPTER 1

INTRODUCTION

Ligninolytic fungi or lignin-degrading fungi have become intensively studied by mycologist throughout the world due to it favorable characteristics over biodegrading bacteria. Their colonization on soil, reproduction by numerous spores and the great metabolic versatility of fungi provides advantages for fungi over bacteria in ease of downstream processing and in handling waste biomass. Ligninolytic enzyme produces by microbial degradation is useful to some extent. But under certain circumstances such as environmental stress, microbial degradation not always occurred at optimum rate. In recent years more interest were put to intensively study alternative source for ligninolytic enzyme. Ligninolytic fungi are major producer of this enzyme, which given the right substrate, fungal degradation of lignocelluloses could be much higher than microbial degradation. Although the results under laboratory condition are compromising, it still not applied largely in the field area.

Over the last 10 years, many species of beneficial fungi have been manipulated for food (e.g. mushrooms), medicines (e.g. Penicillin), biological control, and for the treatment of diseases due to fungal infections. Screening of ligninolytic fungi have also contribute to several biotechnology applications, such as biopulping, biobleaching, and soil bioremediation (Bucke, 1998). Whiteand brown-rot fungi are the most known wood-degrading fungi. White-rot fungi utilize cellulose and hemicelluloses, with higher utilization rate on lignin to carbon dioxide and water. Brown-rot fungi, on the other hand only utilize cell wall components, but leaving the lignin undigested (Highley and Dashek, 1998).

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Lignin degradation mechanism occurs during secondary metabolism. Using P. chrysosporium as a model organism for white-degrading studies, it was shown that when nutrient depletion (nitrogen, carbon or sulphur) occurred in the culture medium, secondary metabolism is triggered. When fungi used up the substrate, the substrate become limited, and lignin degradation begins. Lignin degradation involves extracellular production of Lignin peroxidase (LiP), Manganese peroxidase (MnP) and hydrogen peroxide-generating system. In some species, laccases and cellulase enzymes are also being produced along with haem peroxidases.

Many previous scientists develop laboratory methods to screen and characterize ligninolytic fungi from their natural habitat. These methods have facilitated rapid screening of lignindegrading fungi. The fungi are grown in a suitable culture medium to support mycelia growth. The pure isolates of the ligninolytic fungi are then selected based on their degrading activity over anthraquinone-based dyes such as Remazol Brilliant Blue R (RBBR), Congo Red and Orange II. Temp et al. (1998) have successfully screen for new, powerful lignin-degrading soil fungi in both man-made and natural environments by using this method.

In this research, this method is applied for screening and characterizing the best ligninolytic fungus (fungi) from mixed organic matter collected at agriculture's area. As agricultural sector had contributes large percentage to the economic of this country, this research have seen the potential of future application using the available lignin-degrading fungi. Waste produced in order to get high-yield of the end products from the oil palm for example, can be treated effectively and efficiently by using the degrading ability of ligninolytic fungi. Since many of the previous screening works on ligninolytic enzymes are more concentrated mainly in white rot

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basidiomycetes, there are also possibility of other taxonomic groups capable in the degradation mechanism, such as from the genus Aspergillus and Penicillium.

The main objectives of this study were: 1. To isolate a number of different indigenous species of ligninolytic fungi from three different agricultural waste's samples collected from agricultural sites. 2. To screen fungi based on their dye-degrading abilities on solid agar medium. 3. To select the best ligninolytic enzymes producing fungi based on the enzyme activity assay. 4. To identify the selected fungus isolate by using molecular approaches.

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CHAPTER 2 LITERATURE REVIEW

2.1

Lignin

The lignocellulosic components of plant cells are comprised of cellulose, lignin and hemicellulose. Lignin is complex, heterogeneous, non-stereoregular aromatic polymer composed of phenylpropanoid units (Highley and Dashek, 1998), making it hardly to mineralize it. Lignin binding the cells, fibres and vessels those constitute wood and the lignified elements of plants. In the secondary wall layers of the wood cell wall, cellulose fibres are orientated in parallel chains (Highley and Dashek, 1998), which each layers have different orientation. Hemicellulose bounds to the cellulose fibers by hydrogen bonds. Lignin components are surrounding and interdispersed amongst the cellulosic components. The bond linkages within the lignin polymer make lignin as an ultimate recalcitrant molecule. Degradation of lignocellulose can be achieved using the woodrotting Basidiomycetes, especially the white-rot fungi.

2.2

Ligninolytic fungi

Fungi are natural decomposer by decomposing dead plant matter on the soil surface. These eukaryotes, which are mostly terrestrial, have a diverse group of more than 60,000 known species. The diverse group of these single-celled or multicellular organisms work together to degrade the matter and compost, and obtains food through direct absorption of nutrients throughout the process.

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Ligninolytic fungi are group of fungi that are able to produce extracellular lignin-degrading enzymes. These enzymes provide ligninolytic fungi with the ability to degrade lignocelluloses whereby only relatively few microorganisms can. The wood-rotting Basidiomycetes, called the white-rot fungi (D’Sauza et al., 1999), are most effective in degrade all the components of the cell wall including lignin by using an array of extracellular enzymes. The vegetative fungal hyphae of the fungi cause degradation within the wood structure by penetrating into the wood cells and attacking the lignocellulose polymers.

Several scientific researches had been done previously on ligninolytic fungi species such as Phanerochaete chrysosporium, Coriolus versicolor, Pycnoporus cinnabarinus, and Bjerkandera spp. (D’Sauza et al., 1999). Even the production of lignin-modifying enzymes (LMEs) under different culturing conditions by Ganoderma lucidum ( D’Sauza et al., 1999) were extensively studied despite of it medicinal properties. Studies on the enzymatic composition of the ligninolytic system of white rot fungi Coriolopsis rigida by Saparrat et al. (2002) has provide the information on the oxidative capabilities of this enzyme to degrade wheat straw lignin and both the aliphatic and aromatic fractions of crude oil (Colombo et al., 1996) from contaminated soils. Similarly, the white-rot Lentinus edodes and Pleurotus ostreatus (Ardon et al., 1998) also involve in lignin degradation. The white rot fungi Trametes multicolor (Leitner et al., 2001) that produce the enzyme pyranose oxidase (P2O) is also become well-studied organism due to it significant to the degradation of hardwood, mainly Betula (Leitner et al., 2001) species.

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2.3

Enzymes secreted by ligninolytic fungi

Three out of four classes of extracellular enzymes that have been implicated in lignin degradation are Lignin peroxidases (LiPs), Manganese peroxidases (MnPs) and laccases. The H2O2-generating enzyme glyoxal oxidase (GLOX) (Tien and Kirk, 1984), have been found to limited only to certain species that have been studied previously. The temporal correlation of glyoxal oxidase, peroxidase and oxidase substrates in cultures suggest a close physiological connection between these components. The possibility for production of cellulase enzyme is also there since cellulose component is surrounding by lignin polymer.

2.3.1 Lignin peroxidase (LiP)

Lignin peroxidase (ligninase) plays a central role in the initial degradation of the complex aromatic polymer lignin with this organism. The H2O2-dependent Cα-Cβ cleavage of lignin model compounds has become the key in the discovery of LiP. This model has subsequently shown to catalyze the partial depolymerization of methylated lignin in vitro (Tien and Kirk, 1984). This isozyme is first isolated from the basidiomycete Phanerochaete chrysosporium (Tien and Kirk, 1988). LiP is approximately 37,000 Da in size (Vishal and Frantisek, 2002), and utilize hydrogen peroxide and organic peroxides to oxidize a variety of substrates. All oxidation are dependent on H2O2. Lignin peroxidase oxidizes the aromatic nuclei of substrate by one electron (Cullen and Kersten, 1992). Ligninase production is utilized by nutrient starvation, especially in nitrogen- and carbon-limited cultures. Veratryl alcohol is also very important reducing substrate for ligninase. This enzyme is uniquely characterized by their very low pH optima (Vishal and

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Frantisek, 2002) and much higher redox potentials (>1.3 V) (Haemmerli et al., 1986), other than it ability to oxidize lignin monomers, dimers, and trimers, as well as polycyclic aromatic compounds such as benzopyrene (Haemmerli et al., 1986).

2.3.2 Manganese peroxidase (MnP) MnP is heme-containing enzyme and oxidizes Mn2+ to Mn3+ using H2O2 as oxidant. The enzyme is stimulated by simple organic acids that stabilize the Mn3+ and allow it to oxidize organic compounds including phenolic lignin model compounds (Cullen and Kersten, 1992). The source of H2O2 required as oxidant is glyoxal oxidase (Cullen and Kersten, 1992), under standard ligninolytic conditions. These are including the use of glucose or xylose in the culture media. Unique characteristic of MnP amongst other peroxidases is its principal substrates, which are organic acids such as oxalate, malonate, and lactate. This complex is able to oxidize secondary substrates, such as phenolic compounds, lignin model compounds, high molecular weight chlorolignin (Lackner et al., 1991) and chlorophenols. In the presence of pyrophosphate, the activity of the MnP is enhancing by various chelators such as malonate, oxalate, L-tartrate and methylmalonate (Forrester et al., 1990).

2.3.3 Laccase

This copper-containing enzyme reduces molecular oxygen to water. Both phenolic and nonphenolic aromatic compounds with relatively low oxidation-reduction potentials (D’Sauza et al., 1999) can be degraded by laccases. Nonphenolic substrates with high oxidation-reduction potentials and certain xenobiotics (D’Sauza et al., 1999) can be oxidizing in the presence of low-

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molecular-weight mediators. ABTS (2, 2´-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid) substrates can act as mediators to facilitate the oxidation of non-phenolic compounds. Alternatively, 2, 6-dimethoxyphenol (DMP) and tetramethylbenzidine are also used as substrates to stimulate production of laccases. Among ligninolytic fungi that have been found producing laccases were Pycnoporus cinnabarinus (Eggert et al., 1996), Lepista sordida (Pereira et al., 2004) and Trametes villosa (Polyporus pinsitus or Coriolus pinsitus) (Yaver et al., 1996). The laccase of Trametes versicolor is a blue copper oxidase that catalyses the four-electron reduction of O2 to H2O during its oxidation of phenolics, aromatic amines, ascorbate and methyl cyanides (Cullen and Kersten, 1992). Due to their low specificity, laccase is most important especially for lignin degradation and humification processes (Pereira et al., 2004).

2.3.3 Cellulase Cellulase enzyme is enzyme secreted by fungi during cellulose degradation on wood. Cellulose comprises approximately 45% of dry wood weight, the most abundant renewable organic resource than lignin and hemicellulose. The soft-rot a fungus, Trichoderma reesei (Cullen and Kersten, 1992) is by far the most extensively studied. Most cellulolytic fungi secrete a complex array of degradative enzymes in submerged culture, which can be divided into 3 classes: Endo-1, 4-β-glucanases (EG), Exo-1, 4- β-glucanases (exo-1, 4- β-glucan cellobiohydrolases, CBH) and 1, 4-β-glucosidases (Cullen and Kersten, 1992). All three enzymes have been identified in Phanerochaete chrysosporium and Trichoderma reesei (Cullen and Kersten, 1992).

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2.4

Morphological identification of fungal isolates

Screening of the fungi provides definitive identification of genus and species. Screening can be done by microscopic examination of fungal reproductive structures as well as gross mycelia appearance on the agar plate. The features are including conidial (shape, size, and color), arrangement within the fruiting body, and mycelium (color, size, shape, and texture). Dye decolorization is another method usually applied to facilitate screening of fungi that requires morphological and molecular identification. Dyes are stable, soluble and cheap substrates with high rates of molar extinction and low toxicity, thus offer advantages in ease of handling for a simple, quick and quantitative spectrophotometric assays. Decolorization of dye is directly correlated to its ability to degrade lignin (Vishal and Frantisek, 2002). Glenn and Gold (1983) suggested that the decolorization is a secondary metabolic activity linked to the fungi ligninolytic degradation activity. The extent of color removal varied depending on the dye complexity, nitrogen availability in media, and the ligninolytic activity of the culture. Anthraquinone-based dyes (Vishal and Frantisek, 2002) are structurally similar to the lignin backbone and are thus efficiently decolorized by the white-rot fungi.

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2.5

Molecular identification of fungal isolates by ITS primer

Molecular identification is facilitated by using a set of universal primer known as Internal Transcribed Spacer (ITS) 4 and 5. The primer sequences are as below: ITS 4

(5’- CGTTACTRRGGCAATCCCTGTTG -3’)

ITS 5

(5’- GGAAGTAAAAGTCGTAACAAGG -3’) (White et al., 1990)

ITS primers contain the region that could be amplified for molecular identification of the target fungal isolates. The primer located at the 18S and 28S of the ribosomal RNA (rRNA). ITS primers use the conserved regions of the 18S, 5.8S and 28S ribosomal RNA genes to amplify the non-coding regions between them (White et al., 1990). ITS 4 and 5 are two of the most widely used ITS primers for fungal phylogenetic studies, with expected amplicon sizes of 300 bp and 600 bp, respectively.

The PCR composition for ITS 4 and 5 including PCR buffer, deoxynucleotide triphosphates (dNTPs), ITS 4, ITS 5, Taq DNA Polymerase, DNA template and sterile autoclave distilled water. The final volume of the PCR composition is 25 μl to facilitate the small microcentrifuge tube. The PCR reaction undergo 30 cycles for repeated denaturation, annealing and elongation stages.

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CHAPTER 3

MATERIALS AND METHODS

3.1

Materials

Pineapple's waste samples were collected at Pineapple Farm in Bau District, while oil palm's fruits were provided by Mr. Azis, whereas rubber's leaves and twigs were obtained from the sample collection at Molecular Laboratory of UNIMAS. Main chemicals and materials used, which including Congo Red (Labchem), Internal Transcribed Spacer (ITS) 4 and 5 primers (1st BASE, Malaysia), H2O2 (hydrogen peroxide) (Fluka), 2, 6-dimethoxyphenol (DMP) (Fluka), CMC (carboxymethylcellulose) (Fluka), phenol red (Sigma), veratryl alcohol (Fluka) and all other chemical and material used in this project, unless otherwise stated, were provided by Molecular Laboratory of UNIMAS.

3.2

Methods

3.2.1

Collection of agriculture’s waste samples

Sample of pineapple's leaves, fruits, roots and dried trunks were randomly collected into nylon bags. All samples were air-dried at room temperature before stored in the cold room at -20°C until further used.

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3.2.2

Fungal isolation

In this study, tiny sections were cut from the fruiting body of the growing fungi, and part of wood, fruit, branch and the bark from every sample. Samples were sterilized by amended in 70% ethanol in a beaker for 10 minutes. After the samples dried, the samples were placed onto the Malt Extract Agar (Merck) and Potato Dextrose Agar (PDA) in the Petri dishes. Freshly prepared Potato Dextrose Agar containing (w/v) per liter medium; 200 g of peeled and sliced potatoes, 20 g of dextrose, 2 g of yeast extract (Fluka) and 15 g of agar (Hamburg), top-up to 1000 ml by using sterile distilled water. 0.5 g of antibiotic Streptomycin and Carbenicillin were added into the PDA agar to inhibit bacterial growth. Petri dishes were incubated at room temperature for three days and at inverted position when mycelia growth was detected. Mycelium from 3-daysold cultures were subsequently transferred into fresh PDA plates to obtained the pure cultures. Fungi were maintained on PDA (Merck) agar. Fungi stock cultures were prepared by cutting small section of the mature mycelium from the pure isolates and placed inside universal bottle containing 200 ml of autoclaved distilled water. Stock cultures were freeze-dried by using liquid nitrogen and stored at -80°C.

3.2.3

Preliminary screening of fungi by dye decolorization test

Screening for all isolates of possible ligninolytic fungi were performed on solid minimal media containing per liter of medium (w/v): 2% agar, 0.5 g MgSO4.7H2O, 0.6 g KH2PO4 and 2 g yeast extract. Pure isolates were also grown on PDA agar plates. Plates were inoculated by placing agar plug of mycelium from an actively growing fungal culture on the center of the Petri dishes. The plates were incubated at room temperature for 3 days. Then, 0.5% (w/v) of RBBR and 2%

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Congo Red dyes solution were added after 3 days of incubation into each plate. Observation on the dye-degrading activities was done at interval of 1 to 5 days, based on the clear decolorization halos. The fungal isolates which have greater probability as ligninolytic fungi were selected for further examination. 3.2.4

Morphology identification of fungal isolated

Fungal cultures grown previously at room temperature for 7 days were examined for mycelium colors and growth patterns. The fruiting bodies of the mycelium were also harvested and prestained with acid Fuschin prior to observation by using Leica DME Light Microscope. Permanent slide cultures were prepared by using nail polished.

3.2.5

Enzyme biochemical test in minimal media

Biochemical test was performed according to method by Pereira et al. (2004). Mycelia plugs from seven fungal isolates were transferred into 60 mL of sterile liquid medium, pH 5.5 containing CaCl2, FeSO4.7H2O, (NH4)2HPO4, NaCl, KH2PO4, ZnSO4.7H2O, MgSO4.7H2O, glucose, yeast extract, MnCl2.4H2O, H3BO3, (NH4)6MO7O24.4H2O in 125 mL Erlenmeyers flasks. Flasks were incubated for 21 days at 27°C in the dark without shaking. Liquid cultures were filtered using Whatman No. 1 filter paper, and the filtrates were used for enzymes assay. Laccase assay was determined by using 2, 6-dimethoxyphenol (DMP) as a substrate at 420 nm. Manganese peroxidase was assayed by using phenol red as a substrate and absorbance was reading at 590 nm. Lignin peroxidase was assayed by monitoring oxidation of veratryl alcohol to

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veratraldehyde at A310. Cellulase activity was determined by dinitrosalicyclic acid (DNS) method using glucose as the standard at A540.

3.2.6 3.2.6.1

Molecular identification of fungal isolated DNA Extraction

Mycelia from 3-days-old fungal cultures grown in PDA medium were cut (3 cm2) and frozen in liquid nitrogen. The frozen mycelia were grounded to a powder in a mortar. 0.2 g of the powder was extracted with DNA extraction buffer containing 100 mM Tris-HCl, pH8.0; 10 mM EDTA, pH 8.0 and 1% (w/v) SDS in 1.5 ml microcentrifuge tube and incubated at 65°C before centrifuged at 13,000 rpm for 15 minutes.

Phenol/Chloroform/Isoamyl alcohol (P: C: I)

(25:24:1) was added and centrifuged (twice). After centrifugation, C: I (24:1) was added and centrifuged for 5 minutes. 1/10 volume was precipitated by 3 M Sodium Acetate (NaOAc), pH 5.2, followed by 1 volume of cold absolute ethanol. After centrifugation, the DNA pellet was washed with 70% ethanol (v/v) and centrifuge for 5 minutes before dissolved in 35 μl double distilled H2O, pH 8.0. The DNA was stored at 4°C for subsequent used.

3.2.6.2

Electrophoresis analysis

Agarose gel electrophoresis was carried out using 1% agarose (Promega). For each sample, 5 μl DNA sample was gently mixed with 1 μl of 6X gel loading dye on a piece of Parafilm. The mixture was loaded into the well in the electrode chamber covered with 1X Tris-acetic-acidEDTA (TAE) buffer. Agarose gel electrophoresis was done at 120 V for 50 minutes.

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