UNIVERSITI PUTRA MALAYSIA

BIOETHANOL PRODUCTION USING LIGNOCELLULOSE BIOMASS HYDROLYSATE FROM INDIGENOUS FUNGI FERMENTATION

AHMADU ALI FAROUQ

IB 2012 12

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BIOETHANOL PRODUCTION USING LIGNOCELLULOSE BIOMASS

HYDROLYSATE FROM INDIGENOUS FUNGI FERMENTATION

By

AHMADU ALI FAROUQ

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements for the Degree of Doctor of Philosophy

May 2012

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DEDICATION

This dissertation is dedicated to my parents:

Late Alhaji Ahmadu Gwandu and Late Hajiya A’ishatu Yalli Ahmadu Gwandu

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment

of the requirements for the degree of Doctor of Philosophy

BIOETHANOL PRODUCTION USING LIGNOCELLULOSE BIOMASS

HYDROLYSATE FROM INDIGENOUS FUNGI FERMENTATION

By

AHMADU ALI FAROUQ

May 2012

Chairman: Professor Dzulkefly Kuang Abdullah, PhD

Faculty: Institute of Bioscience

The use of fossil fuel for energy purposes is becoming increasingly unreliable,

skeptical and environmentally unfriendly while energy generated from foods such as

corn and sugarcane is generating crisis of food prices inflation globally. Although,

lignocellulose provides a better alternative source for bioethanol, pretreatment of the

lignocellulose biomass for efficient enzymatic hydrolysis of the substrates is a major

challenge. Pretreatment has been viewed as one of the most expensive processing

steps within the conversion of biomass to fermentable sugar. The use of naturally

sourced native fungi for the pretreatment of lignocellulose biomass materials is thus

a viable alternative devoid of toxic chemicals. In this study, eight native fungi were

isolated from Asian elephant (Elephas maximus) dung sourced locally from the

natural environment. The isolated fungi were identified morphologically and through

the use of molecular assay. All the fungi isolated were further screened for

cellulolytic activities and candidate fungi with best cellulolytic activity, namely, T.

aureoviride strain UPM 09 and F. equiseti strain UPM 09 were selected and used for

pretreatment of lignocellulose biomass materials, namely, rice husk (RH), rubber

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wood saw dust (RW) and oil palm empty fruit bunch (EFB). Prior to pretreatment,

the effect of temperature (30°, 40° and 50°C) on the growth of the fungi was

investigated based on optical density (600nm), dry weight and FPase activity. Solid

state cultivation (SSC) and submerged cultivation (SMC) methods were used for

pretreatment of biomass materials using individual fungi and their consortium.The

effect of fungi on pretreatment of biomass materials was analyzed using X-Ray

diffraction and scanning electron microscopy. Additionally, cellulase enzymes

production was analyzed and the composition of lignocellulose biomass was

determined before and after the pretreatment. Following pretreatment, the best

pretreated biomass material was selected for bioethanol production using

Saccharomyces cerevisiae D5A (ATCC 200062) by Simultaneous Saccharification

and Fermentation (SSF). Ethanol production was analyzed using Gas

Chromatography. The result of the present study shows that T. aureoviride strain

UPM 09 and F. equiseti strain UPM 09 by submerged cultivation (agitated) (SMC)

converted about 60-80% of the biomass substrates to glucose thus exhibiting

pretreatment and saccharification of the lignocellulose biomaterials simultaneously.

GC-MS analysis revealed the presence of volatile hydrocarbons as by-products in the

samples of rubber wood saw dust and oil palm empty fruit bunch. F. equiseti strain

UPM 09 and T. aureoviride strain UPM 09 produced the highest amount of glucose,

reducing sugar with a correspondingly higher amount of enzymes FPase, CMCase,

and beta-glucosidase and protein concentration during submerged cultivation

(agitated). The results of statistical analysis of the pretreated biomass substrates

showed that there was no significant difference (P>0.05) in pretreatment by T.

aureovide UPM 09 and Fusarium equiseti strain UPM 09 either individually or in

consortium using either SSC or SMC against RH, RW or EFB. The lignin and

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hemicellulose reduction was 19.9% and 21.17%, 11.17% and 24.97%, and 31.93%

and 21.77% for RH, RW and EFB, respectively. Therefore, any of the two fungi can

be used for pretreatment purposes either individually or in consortium. Likewise, any

of the pretreatment methods can be used. There was also no significant difference

(P>0.05) in cellulase production between the two fungi (individually or in

consortium) in either SSC or SMC in the three biomass materials used (RH, RW and

EFB). In conclusion, the new native fungi can be used in the pretreatment of the

lignocellulose biomass materials by submerged cultivation or solid state cultivation

using native T. aureoviride strain UPM 09 (JN811061) and F. equiseti strain UPM

09 (JN811063) either individually or in consortium may offer good prospects in the

bioethanol production industry by being cost-effective and environmentally friendly.

This is because the two fungi each individually and in consortium exhibited a

potential for achieving simultaneous pretreatment and hydrolysis of biomass thus

shortening the steps in bioethanol production from three to two and at the same time

the cost of commercial cellulase enzyme will be reduced. In addition, non-volatile

hydrocarbon compounds were produced as by-products by the new native fungi

during pretreatment using submerged cultivation. This study provides the first use of

native fungi isolates of elephant dung for bioethanol production from lignocellulose

biomass in Malaysia.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan ijazah Doktor Falsafah

PENGHASILAN BIOETANOL MENGGUNAKAN HIDROLISAT BIOJISIM

LIGNOSELULOSA DARIPADA FERMENTASI KULAT ASLI

Oleh

AHMADU ALI FAROUQ

Mei 2012

Pengerusi: Professor Dzulkefly Kuang Abdullah, PhD

Fakulti: Institut Biosains

Pengunaan bahan fosil bagi penjanaan tenaga telah semakin meruncing dan tidak

mesra alam. Di samping itu, tenaga yang dihasilkan daripada makanan tumbuhan

seperti jagung dan tebu telah menimbulkan krisis inflasi harga makanan dunia.

Walaupun lignoselulosa menawarkan alternatif yang lebih baik sebagai satu sumber

bioetanol, prarawatan lignoselulosa diperlukan bagi mendapatkan hidrolisis enzim

berkesan terhadap substrat tersebut merupakan satu halangan yang utama.

Prarawatan telah dikenalpasti sebagai salah satu proses termahal di dalam proses

penukaran bahan biojisim kepada gula fermentasi. Penggunaan kulat asli yang

diperolehi secara semulajadi bagi prarawatan bahan biojisim lignoselulosa

merupakan alternatif sesuai tanpa menggunakan bahan kimia yang memudaratkan.

Di dalam penyelidikan ini, lapan kulat asli telah diasingkan daripada najis gajah Asia

((Elephas maximus) yang diperolehi secara tempatan di persekitaran semulajadi.

Kulat yang diasingkan telah dikenalpasti secara morfologi dan juga menggunakan

asai molekul. Kesemua kulat yang diasingkan telah kemudiannya disaring bagi

mengenalpasti aktiviti selulolitik dan calon kulat yang mempunyai aktiviti selulolitik

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terbaik, iaitu, T. aureoviride strain UPM 09 dan F. equiseti strain UPM 09 yang telah

dipilih dan digunakan bagi tujuan prarawatan bahan biojisim lignoselulosa seperti

sekam padi, habuk kayu getah dan buah tandan kosong kelapa sawit. Sebelum

prarawatan, kesan suhu (30°, 40° dan 50°C) kepada pertumbuhan lignoselulosa telah

dikaji berdasarkan ketumpatan optikal (600nm), berat kering dan aktiviti FPase.

Kaedah-kaedah kultivasi keadaan pejal dan kultivasi terendam telah digunakan bagi

prarawatan bahan biojisim bagi setiap lignoselulosa dan konsortiumnya. Kesan kulat

terhadap prarawatan bahan biojisim telah dianalisa menggunakan pembelauan X-Ray

dan mikroskop elektron pengimbas. Di samping itu, penghasilan enzim selulosa telah

dianalisa dan komposisi bahan biojisim lignoselulosa telah ditentukan sebelum dan

selepas prarawatan. Setelah prarawatan, bahan biojisim diprarawat terbaik telah

dipilih bagi penghasilan bioetanol menggunakan Saccharomyces cerevisiae D5A

(ATCC 200062) melalui Saccharification Serentak dan Fermentasi. Penghasilan

bioetanol telah dianalisa menggunakan Gas Kromatografi. Hasil penyelidikan ini

menunjukkan bahawa T. aureoviride strain UPM 09 dan F. equiseti strain UPM 09

melalui kultivasi terendam (diagitasi) menukarkan 60-80% substrat biojisim kepada

glukosa seterusnya mempamerkan prarawatan dan saccharification bahan biojisim

lignoselulosa secara serentak. Analisis GC-MS menunjukkan kehadiran hidrokarbon

volatile sebagai produk sampingan di dalam sampel habuk kayu getah dan tandan

sawit kosong kelapa sawit. F. equiseti strain UPM 09 dan T. aureoviride strain UPM

09 menghasilkan jumlah glukosa tertinggi dengan reducing sugar mengandungi

jumlah enzim FPase, CMCase, dan beta-glucosidase serta kepekatan protein yang

lebih tinggi semasa kultivasi terendam (diagitasi). Hasil analisis statistik terhadap

substrat biojisim diprarawat menunjukkan bahawa tiada perubahan ketara (P>0.05)

di dalam prarawatan oleh T. aureoviride strain UPM 09 dan Fusarium equiseti strain

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UPM 09 sama ada secara inidividu atau konsortium menggunakan sama ada

kultivasi keadaan pejal atau kultivasi terendam terhadap sekam padi, habuk kayu

getah dan buah tandan kosong kelapa sawit. Pengurangan lignin dan hemiselulosa

adalah 19.9% dan 21.17%, 11.17% dan 24.97%, serta 31.93% dan 21.77% bagi

sekam padi, habuk kayu getah dan buah tandan kosong kelapa sawit, masing-masing.

Justeru itu, mana-mana dari dua kulat boleh digunakan bagi tujuan prarawatan sama

ada secara individu atau konsortium. Sehubungan dengan itu, mana-mana kaedah

prarawatan boleh digunapakai. Tiada sebarang perubahan ketara (P>0.05) di dalam

penghasilan selulosa oleh kedua-dua kulat (secara individu atau konsortium) sama

ada melalui kultivasi keadaan pejal atau kultivasi terendam terhadap ketiga-tiga

bahan biojisim yang digunakan. Secara kesimpulan, kulat asli baru boleh

digunapakai bagi tujuan prarawatan bahan biojisim lignoselulosa melalui kultivasi

keadaan pejal atau kultivasi terendam menggunakan T. aureoviride strain UPM 09

(JN811061) dan F. equiseti strain UPM 09 (JN811063) asli secara individu atau

konsortium seterusnya mampu menawarkan prospek baik kepada industri

penghasilan bioetanol secara lebih ekonomi dan mesra alam. Ini disebabkan kedua-

dua kulat masing-masing secara individu dan konsortium mempamerkan potensi

meningkatkan prarawatan dan hidrolisis biojisim serentak, justeru memendekkan

langkah penghasilan bioetanol dari tiga ke dua, dan sekaligus menjimatkan masa

serta kos enzim selulosa komersial. Tambahan pula, sebatian hidrokarbon tidak

volatile telah dihasilkan sebagai produk sampingan oleh kulat asli baru semasa

prarawatan melalui kaedah kultivasi terendam. Penyelidikan ini menyumbang

kegunaan pertama kulat asli yang diasingkan dari najis gajah bagi tujuan penghasilan

bioetanol daripada biojisim lignoselulosa di Malaysia.

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ACKNOWLEDGEMENTS

All thanks are due to the Almighty Allah in His mercy for making this journey

possible. I wish to express my sincere gratitude and appreciation to my supervisor

Prof Dr. Dzulkefly Kuang Abdullah for his guidance, patience and invaluable

contribution throughout the course of this work. This gratitude and appreciation is

extended to my co-supervisors Prof. Madya Dr. Foo Hooi Ling and Prof. Madya Dr.

Norhafizah Abdullah for their supervision and contributions in this work.

I am very grateful to Alhassan M. Alhassan for the academic discourse, cooperation,

assistance, friendship and support i enjoyed from him in the course of this study. I

wish to thank Mr. Mirsasan Mirpour and Miss Forough K. Nazarpour, with whom

we worked together in the Laboratory of Industrial Biotechnology at the Institute of

Bioscience (IBS), for their cooperation. I am also especially grateful to Pn. Zainura

Mat Hussein, Encik Ahmad Adlie Shamsuri and other members of staff of our

laboratory and other laboratories in IBS and other faculties: Electron Microscopy

Unit and Laboratory of Marine Science and Biotechnology especially Mr. Perumal

Kuppan for the cooperation and support I enjoyed from them in the course my

experiments at the Institute of Bioscience.

I am very grateful to Abdoulie Ceesay of IBS for his cooperation, assistance,

friendship, support and academic discourse I enjoyed from him in the course of this

study. I am also very grateful to Dr. Faruk Bande for formatting the thesis.To many

others, whose names are too numerous to mention and to avoid the risk of making

some important omissions here, I am very grateful. Finally, I am most grateful to my

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brother Usman Ahmed for financial, spiritual and moral support; and to my family,

particularly my wife Mrs. Fatima Yusuf, my children Hauwa’u Maijidda Farouq,

Halimat Sadiya Farouq and Ahmad Aslam Farouq for their understanding, patience

and moral support.

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I certify that a Thesis Examination Committee has met on 23rd May,2012 to conduct

the final examination of Ahmadu Ali Farouq on his thesisentitled “Bioethanol

Production Using Lignocellulosic Biomass Hydrolysate from Indigenous Fungi

Fermentation ’’ in accordance with the Universities and University colleges Act 1971

and Constitution of Universiti Putra Malaysia [P.U.(A)106] 15 March 1998. The

Committee recommends that the student be awarded the Doctor of Philosophy.

Members of the Thesis Examination Committee were as follows:

Y. Bhg. Prof. Dr. Gwendoline Ee Cheng Lian

Chemistry Department

Faculty of Science

Universiti Putra Malaysia

(Chairman)

Assoc. Prof. Dr. Rosfarizan Binti Mohamad

Department of Bioprocess Technology

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Internal Examiner)

Assoc. Prof. Dr. Muskhazli b Mustapha

B iology Department

Faculty of Science

Universiti Putra Malaysia

(Internal Examiner)

Assoc. Prof. Dr. Ratna R. Sharma-Shivappa

Biological and Agricultural Engineering D.S Weavers labs,

North Carolina State University, Campus box 7625,

North Carolina, USA.

(External Examiner)

SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

.

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for Doctor of Philosophy. The members of

the Supervisory Committee were as follows:

Dzulkefly Kuang Abdullah, PhD

Professor

Faculty of Science

Universiti Putra Malaysia

(Chairman)

Foo Hooi-Ling, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Member)

Norhafizah Bt. Abdullah, PhD

Associate Professor

Faculty of Engineering

Universiti Putra Malaysia

(Member)

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is

not concurrently, submitted for any other degree at Universiti Putra Malaysia or at

any other institution.

_______________________

AHMADU ALI FAROUQ

Date:

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TABLE OF CONTENTS

Page

DEDICATION ii

ABSTRACT iii

ABSTRAK vi

ACKNOWLEDGEMENTS ix

APPROVAL xi

DECLARATION xiii

LIST OF TABLES xvii

LIST OF FIGURES xviii

LIST OF ABBREVIATIONS xxii

CHAPTER

1.0 INTRODUCTION 1

2.0 LITERATURE REVIEW 7

2.1 Renewable Energy (RE) in Malaysia 8

2.2 Pretreatment 15

2.2.1 Methods of pretreatment 19

2.2.1.1 Physical Pretreatment 19

2.2.1.2 Physico-Chemical Pretreatment 20

2.2.1.3 Chemical Pretreatment 22

2.2.1.4 Organoslov Pretreatment 26

2.2.1.5 Ionic Liquid Pretreatment 27

2.3 Lignocelluloses 28

2.3.1 Composition of lignocelluloses residues 29

2.3.2 Biodegradation of lignocelluloses residues 31

2.3.2.1 Cellulose biodegradation 32

2.3.2.2 Hemicellulose biodegradation 33

2.4 Coprophilous fungi 34

2.5 Biological Pretreatment 35

2.6 Bioconversion of lignocellulose into byproducts 37

3.0 MATERIALS AND METHODS 40

3.1 Materials 40

3.1.1 Microorganism 40

3.2 Collection of Asian Elephant (E. maximus) Dung Samples 40

3.3 Sample Collection Site 41

3.4 Preparation of Elephant Dung 42

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3.5 Inoculation 42

3.6 Morphological Identification and Characterization of Fungi 42

3.6.1 Sub culturing to obtain pure cultures of fungi 43

3.7 Molecular Identification of Native Fungi 43

3.7.1 Nucleic Acid (DNA) Extraction, Concentration and Purity 43

3.7.2 PCR Amplification and Sequencing 44

3.7.3 Electrophoresis 45

3.7.4 Molecular Sequence Analysis 45

3.8 Qualitative Screening of Native Fungi for Lignocellulolytic Activity 45

3.8.1 Screening of Native Fungi for cellulolytic Activity 45

3.8.2 Screening of Native Fungi for Lignolytic Activity 46

3.9 Effect of temperature on Growth Condition of the Native Fungi and FPase

Activity 47

3.10 Rubber Wood Saw dust, Rice Husk and Oil Palm Empty Fruit Bunch 48

3.11 Preparation of Rubber Wood Saw Dust, Rice Husk and Oil Palm Empty Fruit

Bunch 48

3.12 Determination of the Composition of Rubber-wood Saw dust, Rice husk and

Oil palm empty fruit bunch 49

3.12.1 Compositional Analysis 49

3.14 Spectroscopic Analysis 53

3.15 X-ray Diffraction 53

3.16 Scanning Electron Microscopy (SEM) 54

3.17 Pretreatment 54

3.17.1 Submerged Cultivation 55

3.17.2 Solid State Cultivation (SSC) 56

3.18 Enzyme Hydrolysis 57

3.19 Determination of Reducing Sugar by Dinitrosalicylic Acid (DNS) Method 58

3.20 Determination of Glucose 58

3.21 Filter Paper Assay for Saccharifying Cellulase (FPase) 59

3.22 Carboxymethyl Cellulase Assay (CMCase) For Endo-β-1, 4-glucanase 60

3.23 β-glucosidase (BGL) Assay 60

3.24 Determination of by-products from pretreated biomass materials 61

3.25 Production of Ethanol 61

3.26 Gas Chromatography (GC) 63

3.27 Statistical Analysis 64

4.0 RESULTS AND DISCUSSION 65

4.1 Native Fungi isolated from Elephant dung 65

4.2 PCR Amplified Fragments of Genomic DNA of Native Fungi 65

4.3 Phylogenetic tree of the Native Fungi 67

4.4 Screening of Native fungi in this study for cellulolytic activity 68

4.5 Effect of Temperature on Growth Condition of the Native Fungi and Enzyme

Production (FPase) 70

4.6 Composition Profile of Untreated and Treated Lignocellulose

biomass materials 72

4.7 Effect of Pretreatment on the degradation of rice husk(RH), rubber wood saw

dust (RW) and empty fruit bunch (EFB). 74

4.7.1 Effect of Pretreatment 74

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4.9 Scanning Electron Microscopy 88

4.10 Spectroscopy analysis of pretreated sample 90

4.10.1 X-ray Diffraction 90

4.11 Enzymatic Hydrolysis 93

4.12 Ethanol Production 94

4.13 By-products obtained from pretreated EFB and RW using submerged

cultivation SMC by native fungi 96

5.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS FOR FUTURE

RESEARCH 101

5.1 Summary 101

5.2 Conclusion 101

5.3 Recommendations for future research 103

REFERENCES 104

APPENDICES 117

BIODATA OF STUDENT 144

LIST OF PUBLICATIONS 145