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UNIVERSITI PUTRA MALAYSIA

BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE-co-3- HYDROXYVALERATE) COPOLYER FROM ORGANIC ACIDS USING

Comamonas sp. EB172

MOHD RAFEIN BIN ZAKARIA @ MAMAT

FBSB 2011 17

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BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE-co-3-

HYDROXYVALERATE) COPOLYER FROM ORGANIC ACIDS USING

Comamonas sp. EB172

By


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

in Fulfilment of the Requirements for the Doctor of Philosophy

August 2011

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DEDICATION

This work is dedicated to my lovely family, who has always been at my side and

given me the encouragement and support that carries me through my study.

Thanks for their unending love and care to me.

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Abstract of thesis presented to Senate of Universiti Putra Malaysia in fulfilment of

the requirement for the Doctor of Philosophy

BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE-co-3-

HYDROXYVALERATE) COPOLYMER FROM ORGANIC ACIDS USING

Comamonas sp. EB172

By


August 2011

Chairman: Professor Mohd Ali Hassan, PhD

Faculty: Faculty of Biotechnology and Biomolecular Sciences

Petroleum-based plastics like polypropylene (PP) and polyethylene (PE) are common

plastics used in our daily life. Despite having good thermal and mechanical

properties which make these plastics suitable for wide range applications, they are

non-biodegradable and their accumulation has resulted in environmental concern by

consumers all round the world. Polyhydroxyalkanoates (PHAs) have been

recognized as suitable candidates in replacing partially or completely petroleum-

based plastics since they share similar thermal and mechanical properties. Moreover,

the biodegradability aspect of PHA makes these polymers to be more desirable and

environmental friendly. Nevertheless, the total PHAs production cost is more

expensive in comparison to conventional plastics, a factor that limits the production

of these materials in large scale. The use of a locally- isolated bacterial species that

can convert organic acids derived from anaerobically treated palm oil mill effluent

(POME) with an efficient fermentation process will help to reduce the total PHA

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production cost. In this study, isolation and screening of PHA producing bacterium

was performed by using Nile Blue A staining method. The stained colonies

containing PHA exhibited pink fluorescent colour when exposed under ultra violet

(UV) light.

The PHA producing bacterium obtained from digester- treated POME was

characterized by polyphasic taxonomic approach. The cells were rod-shaped, Gram-

negative, non-pigmented, non-spore-forming and non-fermentative. Phylogenetic

analyses using the 16S rRNA gene sequence showed that the isolate was clustered in

the genus of Comamonas. Thus Comamonas sp. EB172 is designated for the locally

isolated bacterial species. Its closest neighbours are the type strains Comamonas

terrigena (96.8%), Comamonas koreensis (93.4%), Comamonas composti (92.9%)

and Comamonas kerstersii (91.1%). Comamonas sp. EB172 was clearly

distinguished from all of the existing Comamonas species using phylogenetic

analysis, fatty acid composition and a range of physiological and biochemical

characteristics. The G+C content of the genomic DNA was 59.1 mol%. It is evident

from the genotypic, phenotypic data and ability to produce PHA that Comamonas sp.

EB172 represents a new species in the genus Comamonas (GeneBank accession no.

EU847238).

Based on the types of carbon sources tested in shake flasks experiments, poly(3-

hydroxybutyrate) [P(3HB)] homopolymer and poly(3-hydroxybutyrate-co-3-

hydroxyvalerate) [P(3HB-co-3HV)] copolymer were produced by Comamonas sp.

EB172 using single and mixed carbon sources. Poly(3-hydroxyvalerate) P(3HV)

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incorporation in the copolymer was obtained when propionic and valeric acid were

used as precursors. Incorporation of 3HV fractions in the copolymer varied from 45

to 86 mol% when initial pH of the medium was regulated. In fed-batch cultivation,

organic acids derived from anaerobically treated POME which consist mainly acetic,

propionic and butyric acids are shown to be suitable carbon sources for

polyhydroxyalkanoate (PHA) production by Comamonas sp. EB172. The number

average molecular weight (Mn) of P(3HB-co-3HV) copolymer produced by the strain

was in the range of 153 to 412 kDa with polydispersity index (Mw/Mn) in the range of

2.2 to 2.6. Incorporation of higher 3HV units improved the thermal stability of

P(3HB-co-3HV) copolymer. The CDW (14.6 g/L) and the Mn (838 x 103 Da) of

P(3HB-co-3HV) copolymer reached the highest when supplemented with yeast

extract during the fermentation period. Tensile strength, elongation to break and

Young`s modulus of the copolymer containing 6-8 mol% P(3HV) were recorded at

12-15 MPa, 160-710% and 0.19-0.34 GPa respectively. Thus the newly isolated

bacterium Comamonas sp. EB172 is shown to be a suitable candidate for PHA

production using organic acids from POME as a renewable and alternative raw

material.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Doktor Falsafah

BIOSINTESIS POLI(3-HIDROKSIBUTIRAT-co-3-HIDROKSIVALERAT)

KOPOLIMER DARI ASID ORGANIK OLEH Comamonas sp. EB172

Oleh


Ogos 2011

Pengerusi: Profesor Mohd Ali Hassan, PhD

Fakulti: Fakulti Bioteknologi dan Sains Biomolekul

Plastik berasaskan petrolium seperti polipropilena (PP) dan politetilena (PE) ialah

plastik biasa digunakan dalam kehidupan harian kita. Walaupun mempunyai sifat-

sifat mekanikal dan terma yang baik membuatkan plastik ini sesuai dalam pelbagai

aplikasi, bagaimanapun plastik ini tidak terbiodegradasi dan pengumpulannya telah

menimbulkan keprihatinan terhadap alam sekitar oleh pengguna plastik di seluruh

dunia. Polihidroksialkanoat (PHA) telah di kenal pasti sebagai calon yang sesuai

dalam menggantikan sebahagiannya atau sepenuhnya plastik berasaskan petrolium

kerana ia berkongsi sifat-sifat mekanikal dan terma yang sama. Tambahan pula,

keterbiodegredan PHA membuat polimer ini lebih diperlukan dan mesra alam. Walau

bagaimanapun, jumlah kos pengeluaran PHA lebih mahal berbanding dengan plastik

konvensional yang menghadkan pengeluaran bahan-bahan ini dalam skala besar.

Penggunaan pencilan bakteria baru yang boleh menukar asid-asid organik berasal

daripada POME yang terawat serta proses penapaian yang cekap akan dapat

membantu mengurangkan jumlah kos pengeluaran PHA. Dalam kajian ini, pencilan

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dan saringan bakteria yang menghasilkan PHA telah diusahakan dengan

menggunakan kaedah pewarnaan Nile Blue A. Koloni-koloni yang mengandungi

PHA dan diwarnakan akan mempamerkan warna pendarfluor merah jambu apabila

didedahkan di bawah cahaya ultra ungu (UV).

Bakteria yang menghasilkan PHA disaring dari loji rawatan POME, telah dikaji

melalui pendekatan taksonomi. Sel-sel yang diperolehi adalah berbentuk rod, Gram-

negatif, tidak berpigmen, tidak berspora dan tidak terfermentasi. Analisis filogenetik

menggunakan jujukan gen RNA ribosom 16S menunjukkan bahawa bakteria yang

disaring ditempatkan dalam kelompok genera Comamonas. Dengan itu bakteria

terpencil di namakan sebagai Comamonas sp. EB172. Tetangganya yang terdekat

adalah dari jenis Comamonas terrigena (96.8%), Comamonas koreensis (93.4%),

Comamonas composti (92.9%), dan Comamonas kerstersii (91.1%). Comamonas.

Bakteria terpencil Comamonas sp. EB172 jelas dibezakan dari semua jenis

Comamonas yang ada dengan menggunakan analisis filogenetik, komposisi asid

lemak dan pelbagai ciri-ciri fisiologi dan biokimia. Kandungan G+C dari genom

DNA adalah 59.1 mol%. Dari data genotipik, fenotipik dan kemampuan untuk

menghasilkan polihidroksialkanoat membuktikan Comamonas sp. EB172 merupakan

bakteria baru dalam genera Comamonas (GeneBank akses no.EU847238).

Berdasarkan jenis sumber karbon yang diuji dalam eksperimen kelalang goncang,

homopolimer poli(3-hidroksibutirat) [P(3HB)] dan kopolimer poli(3-hidroksibutirat-

co-3-hidroksivalerat) [P(3HB-co-3HV)] dihasilkan oleh Comamonas sp. EB172

menggunakan sumber karbon tunggal dan campuran. Penggabungan poli(3-

hidroksivalerat) P(3HV) pada kopolimer diperolehi apabila asid propionik dan asid

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valerik digunakan sebagai prekursor. Penggabungan unit 3HV pada kopolimer

berbeza-beza dari 45 hingga 86 mol% apabila pH awal media itu dikawal. Dalam

fermentasi suapan-sesekelompok menggunakan asid organik dari rawatan loji

rawatan POME yang terdiri daripada asid asetik, propionik dan butirik membuktikan

ia adalah sumber karbon yang sesuai bagi penghasilan polihidroksialkanoat (PHA)

oleh Comamonas sp. EB172. Jumlah purata berat molekul (Mn) kopolimer P(3HB-

co-3HV) yang dihasilkan oleh Comamonas sp. EB172 masing-masing berada di

lingkungan 153-412 kDa dengan indeks polidispersiti (Mw/Mn) di lingkungan 2.2-2.6.

Penggabungan unit 3HV lebih tinggi dalam kopolimer (3HB-co-3HV) meningkatkan

kestabilan terma poliester. Berat sel kering (14.6 g/L) dan berat molekul (838 x 103

Da) kopolimer P(3HB-co-3HV) mencapai tahap tertinggi apabila dibekalkan dengan

ekstrak ragi (YE) semasa proses fermentasi. Kekuatan ketegangan, pemanjangan

pada tahap putus dan Young`s modulus kopolimer yang mengandungi (3HV) dari

lingkungan 6-8 mol% yang diperolehi dicatat masing-masing sekitar 12-15 MPa,

160-710% dan 0.19-0.34 GPa. Dengan itu pencilan bakteria baru Comamonas sp.

EB172 adalah bakteria yang sesuai untuk penghasilan PHA menggunakan POME

sebagai sumber karbon alternatif baru dan diperbaharui.

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ACKNOWLEDGEMENTS

Alhamdulillah, great thanks to Allah for His mercy and guidance, I have completed

my thesis as required. I would like to express my gratitude to my main supervisor,

Prof. Dr. Mohd Ali Hassan, co-supervisors, Assoc. Prof. Dr. Suraini Abd-Aziz, Dr.

Farinazleen Mohamad Ghazali and Prof. Dr. Yoshihito Shirai from Kyushu Institute

of Technology, Japan, for their dedicated effort, guidance and encouragement

throughout the research. Their experiences are valuable as guidance to my successful

research.

To my parent, I would like to say that without your sacrifice, encouragement and

understandings I may not be able to complete this study. To my wife, Ros, your

support and undying love taught me about life, discipline and working hard towards

our goal. To my daughter Siti Aisyah, you presence in my life is so meaningful and

taught me to be an ambitious man. Not forgetting to all my friends, I would like

specially say thank you to Dr. Hidayah, Dr. Phang, Dr. Nor `Aini, Dr. Helmi, Dr.

Azhari, Dr. Alawi, Mior Ahmad Khushairi, Noor Azman, and Zulkhairi, for their

sharing a joyful moment with me. Words could not express my gratitude to all of

you.

Last but not least, special thanks to our collaborator from FELDA Serting Hilir palm

oil mill, Mr. Omar Bin Atan for his assistance and guidance during my study period.

My seniors, juniors and lab assistants, Shamzi, Dr. Meisam, Nor Asma, Dr. Mumtaz,

Yee, Nadia and Matun for their support, co-operation, sharing ideas and guidance to

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me. Million thanks to lab officer Mr. Rosli for his technical assistance in the

Microbiology and Bioprocess Laboratory.

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I certify that an Examination Committee has met on 24th

August 2011 to conduct the

final examination of Mohd Rafein Bin Zakaria @ Mamat on his Doctor of

Philosophy thesis entitle “Biosynthesis of Poly(3-Hydroxybuyrate-co-3-

Hydroxyvalerate) Copolymer from Organic Acids by Locally Isolated Bacterium

Isolate EB172” in accordance with the Universities and University Colleges Act

1971 and the Constitution of the 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 Examination Committee were as follows:

Arbakariya Ariff, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Norhani Abdullah, PhD

Professor



(Internal Examiner)

Siti Mazlina Mustapa Kamal, PhD

Associate Professor

Faculty of Engineering


(Internal Examiner)

Virendra Swarup Bisaria, PhD

Professor

Department of Biochemical Engineering and Biotechnology

Indian Institute of Technology, Delhi

(External Examiner)

_______________________________

BUJANG KIM HUAT, PhD

Professor and Deputy Dean

School of Graduate Studies


Date:

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

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

of the Supervisory Committee were as follows:

Mohd Ali Hassan, PhD

Professor



(Chairman)

Suraini Abd-Aziz, PhD

Associate Professor



(Member)

Farinazleen Mohamad Ghazali, PhD

Faculty of Food Science and Technology


(Member)

Yoshihito Shirai, PhD

Professor

Graduate School of Life Science and Systems Engineering

Kyushu Institute of Technology, Japan

(Member)

_______________________________

BUJANG KIM HUAT, PhD Professor and Dean

School of Graduate Studies


Date:

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DECLARATION

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

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

concurrently, submitted for any other degree at Universiti Putra Malaysia or at any

other institution.

___________________________________

MOHD RAFEIN ZAKARIA @ MAMAT

Date: 24th

August 2011

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TABLE OF CONTENTS

Page

DEDICATION ii

ABSTRACT iii

ABSTRAK v

ACKNOWLEDGEMENTS vii

APPROVAL ix

DECLARATION Xi

LIST OF TABLES Xv

LIST OF FIGURES xvii

LIST OF ABBREVIATIONS xix

CHAPTER

1.0 INTRODUCTION 1

1.1 Objectives 4

2.0 LITERATURE REVIEW

2.0 Polyhydroxyalkanoate (PHA) 5

2.1 Biosynthesis of P(3HB-co-3HV) Copolymer 9

2.1.1 P(3HB-co-3HV) Copolymer Biosynthethic Pathway 10

2.2 Factors Affecting P(3HB-co-3HV) Copolymer Production 16

2.2.1 Microorganism 17

2.2.2 Carbon sources 18

2.2.3 pH 21

2.2.4 Nitrogen Sources 22

2.2.5 Oxygen Requirement 24

2.3 Recovery od Polyhydroxyalkanoates 25

2.4 Thermal and Mechanical Properties of P(3HB-co-3HV)

Copolymer

27

2.5 General Applications of Polyhydroxyalkanoates 30

3.0 MATERIALS AND METHODS

3.1 Source of Sludge 32

3.2 Growth and Production Medium 32

3.3 Shake Flask Experiment 36

3.4 Analytical Procedures 37

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4.0 ISOLATION AND SCREENING OF

POLYHYDROXYALKANOATE ACCUMULATING

BACTERIUM

4.1 Introduction 48

4.2 Materials and Methods 50

4.3 Results and Discussion 55

4.3.1 Isolation and Screening of PHA Accumulating

Bacterium

55

4.3.2 Pre-identification of Isolated Strain by BIOLOG

System

56

4.3.3 Shake Flask Experiments 57

4.4 Conclusion 61

5.0 IDENTIFICATION OF ISOLATED BACTERIUM

USING POLYPHASIC TAXONOMIC APPROACHES

5.1 Introduction 62



5.3.1 Morphological and Biochemical tests 68

5.3.2 16S rRNA Sequence 71

5.3.3 DNA-DNA relatedness, G+C content and Cellular

Fatty Acids Analyses

73

5.3.4 Growth and Production of Isolate EB172 76

5.3.5 Effect of C/N Ratio on the Growth and PHA

Accumulation

78

5.3.6 Fed-batch Cultivation Using pH-stat Continuous

Feeding of Mixed Acids Derived from POME

79

5.4 Conclusion 82

6.0 CHARACTERIZATION OF GROWTH AND

POLYHYDROXYALKANOATE ACCUMULATION

6.1 Introduction 83



6.3.1 Effect of Propionic and Valeric Acid Concentration 89

6.3.2 Effect of Inorganic and Organic Nitrogen Sources 92

6.3.3 Effect of Initial Medium pH 95

6.3.4 Effect of Aeration 97

6.3.5 Effect of C/N and Organic Nitrogen on the P(3HB-

co-HV) copolymer production

100

6.3.5.1 Cell Biomass and PHA Production 100

6.3.5.2 PHA Composition and Tm 104

6.3.5.3 Molecular Weight and Mechanical Properties 105

6.4 Conclusion 108

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7.0 CHARACTERIZATION OF POLY(3-

HYDROXYBUTYRATE-co-3-HYDROXYVALERATE)

COPOLYMER

7.1 Introduction 109



7.3.1 Biosynthesis of PHA Through One Step Cultivation 114

7.3.2 Fed-batch Cultivation of Isolate EB172 120

7.3.3 Gel permeation chromatography (GPC) analysis 122

7.3.4 Thermal Properties of P(3HB-co-3HV) Copolymer

Produced by Isolate EB172

123

7.4 Conclusion 126

8.0 CONCLUSION AND SUGGESTIONS FOR FUTURE

RESEARCH

127

REFERENCES

132

APPENDICES 143

BIODATA OF STUDENT 166

BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE-co-3-HYDROXYVALERATE) COPOLYER FROM ORGANIC ACIDS USINGComamonas sp. EB172ABSTRACTTABLE OF CONTENTSCHAPTER 1CHAPTER 2CHAPTER 3CHAPTER 4 CHAPTER 5CHAPTER 6CHAPTER 7CHAPTER 8REFERENCESAPPENDICESAPPENDIX 1 COPY OF ACEPTANCE LETTERAPPENDIX 1 PUBLICATIONAPPENDIX 2 COPY OF ACCEPTANCE LETTERAPPENDIX 2 PUBLICATIONPolyhydroxyalkanoate production from anaerobically treated palm oil mill effluent by new bacterial strain Comamonas sp. EB172AbstractIntroductionMaterials and methodsBacterial strain and cultivationMorphology and taxonomical studiesIdentification and 16S rRNA gene sequencingPhylogenetic analysis of 16S rRNA gene sequenceAnalytical procedures

Results and discussionMorphological and taxonomical identificationGrowth and polyhydroxyalkanoates production by Comamonas sp. EB172Effect of C/N ratio on the cell growth and PHA accumulationFed-batch cultivation using pH-stat continuous feeding of mixed organic acids derived from POME

ConclusionAcknowledgmentsReferences

APPENDIX 3 COPY OF ACCEPTANCE LETTERAPPENDIX 3 PUBLICATIONBiosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer from wild-type Comamonas sp. EB172IntroductionMaterials and methodsBacterial strainPHA biosynthesis by one-step cultivationFed-batch cultivation of Comamonas sp. EB172 using organic acids from POMEAnalysis of polymer propertiesGas chromatographyPHA extraction film preparationGel permeation chromatography (GPC)Thermogravimetry analysis (TGA)Differential scanning calorimetry (DSC)

Results and discussionBiosynthesis of PHA during one-step cultivationFed-batch cultivation of Comamonas sp. EB172Gel permeation chromatography (GPC)Thermal properties of P(3HB-co-3HV) copolymer from Comamonas sp. EB172

ConclusionsAcknowledgmentReferences

BIODATA OF STUDENT

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