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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