SINGLE-STAGE BIOSYNTHESIS OF POLY(3- HYDROXYBUTYRATE...

SINGLE-STAGE BIOSYNTHESIS OF POLY(3-

HYDROXYBUTYRATE-co-4-

HYDROXYBUTYRATE) USING Cupriavidus sp.

USMAA1020 VIA MIXED-SUBSTRATE

CULTIVATION STRATEGY

HUONG KAI HEE

UNIVERSITI SAINS MALAYSIA

2017

SINGLE-STAGE BIOSYNTHESIS OF POLY(3-

HYDROXYBUTYRATE-co-4-

HYDROXYBUTYRATE) USING Cupriavidus sp.

USMAA1020 VIA MIXED-SUBSTRATE

CULTIVATION STRATEGY

by

HUONG KAI HEE

Thesis submitted in fulfillment of the requirements

for the degree of

Doctor of Philosophy

March 2017

ii

ACKNOWLEDGEMENT

Everyone who has carried out research and written a thesis experiences the

challenges and difficulties that come in various forms. This thesis is the result of my

work at USM over the last five years. Upon completing the work and thesis, I am

exposed to an environment which boosted up my personal growth, learned what does

it means and how to be a competent researcher. In such a tough time which

eventually marks a significant milestone in my life, I have been taught, inspired and

encouraged by many people, both inside and outside USM. It is such a delightful

moment to introduce these awesome people who have accompanied me along the

journey in this glorious acknowledgement.

I would like to express my profoundest and deepest gratitude to my beloved

supervisor, Prof. Dr. Amirul Al-Ashraf Abdullah. Thank you for trusting me, giving

me freedom to pursue my ideas, and providing me a wonderful workspace to conduct

my research. You are always full of enthusiasm, inspiration, passion and ideas. Your

critical supervision has not only shaped my scientific attitude but also taught me to

view things from different perspectives. It is such a privilege to have you as my

scientific partner. Thank you for being an excellent role model as a responsible

researcher. Your influence does not end here but will definitely enlighten me in

future. I would also like to acknowledge my co-supervisor Assoc. Prof. Dr. Ahmad

Ramli Mohd Yahya as well for his priceless opinions which have deepened my

knowledge in biochemical engineering. I appreciate every single comment and

advice offered by him throughout this research. I would like to take this

opportunity to show my sincere appreciation to all of my lab members who have

been great companion of mine, along with their continuous support and

encouragements. The token of appreciation goes to my

seniors especially Dr. Hema, Dr. Shantini, Dr. Vigneswari, Solehah, Sharmini,

Hezreen, Fahima, Zuriani, Nik Murniati, Muzaiyanah, Faezah, Rennukka and

Hemalatha. Special thanks to my labmates Syafiq, Azuraini, Shazwani, Ain, Nina,

Azura, Iszatty, Sheeda, Hafini, Tse Kiun, Aliaa, Fatin, Hanim, Hani, Aida, Thana

and Amellia. I am grateful with all the happiness, sadness, discussions, learning and

of course the food and movie that we shared together. I have also been blessed with

my friends, Shakilla, Adilah, Asyikin, Thina, Mei Suen, Jen Yang, Qiu Ting, Kee

Shin, Siew Wen, Bee Yin, Xin Yu, Yea Lu, Dr. Anne, Dr. Meng Kiat and Janet Teng

from other research groups who are keen to share their life and lab experience with

me.

I owe my gratefulness to some technical staff that assisted me in handling

various equipments. They are Ms. Jamilah, Mr. Johari, Mr. Masrul and Mr. Rizal

from microscopy unit in School of Biological Sciences; Mr. Mustaqim from X-ray

unit in School of Physics; Mr. Nizam and Mr. Zahari from NMR unit in School of

Chemical Sciences; Mr. Anuar for DSC analysis in IPharm. I would like to thank Dr.

Teh Chin Hoe from Bruker who has contributed ideas and advices in my NMR

studies. I would like to extend my gratitude to Syafiq, Ganesh and Muhidhir who

helped a lot in bioprocess unit, IPharm. My appreciation also goes to previous and

current Executive Directors of IPharm, Prof. Dato Dr. Mohamed Isa Abdul Majid,

Prof. Dr. Habibah Abdul Wahab and Assoc. Prof. Dr. Mohamed Ibrahim Noordin;

management staff as well in IPharm, including Mrs. Wan Shahirah, Mrs Saaidah, Mr.

Haifz, Mr. Zulfadli, Mrs. Azira, Ms. Khadijah and Ms. Nusaibah.

Throughout these years, I am grateful to MyPhD scholarship from Ministry

of Higher Education (2011-2014) and USM graduate assistance scheme (2015-2016)

from Institute of Postgraduates Studies, USM which funded my tuition fee and living

allowance. It is also my honor to be one of recipients of Science and Technology

Research Grant (STRG) from Malaysia Toray Science Foundation (MTSF) in 2013

which financially supported part of my research.

The acknowledgement wouldn’t be complete without giving thanks to my

family and friends. I am indebted to my family members for their unconditional love,

constant and continuous support. We are distanced but our relationship doesn’t easily

fade off. Dad, mom, brother and sister, your presence in my life has always made me

feel so significant and strong enough to face my life obstacles. I am also thankful to

my friends who understand, respect and accept me for who I am. This is especially

pointed to Leonard Lu and his wife Siew Hiong who always lend their ears and

hands. Their kind words have always inspired and constantly pushed me to strive for

the best. I appreciate Dr. Lai Kee Huong and Sumithda Lim for their willingness to

share their expertise which has also contributed to the completion of my research

project. There are many others whom I may have left out but I sincerely thank all of

them from the bottom of my heart.

Above all, I thank God for His grace who gave me strength and perseverance

to complete my doctoral study. Pursuing higher education doesn’t means I am so

significant that God is no more relevant and needed in my life. In fact, throughout

this journey I come to realize how small I am and how significant He is in this

broken world. I have always kept remembering the bible verse in Proverbs 9:10:

“The fear of the LORD is the beginning of wisdom, and knowledge of the Holy One

is Understanding” which reminds me that everything comes from him and I have to

be humble before Him. Thank you Lord for being the way, the truth and the life.

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENT ii

TABLE OF CONTENTS iii

LIST OF TABLES vii

LIST OF FIGURES x

LIST OF SYMBOLS AND ABBREVIATIONS xiv

ABSTRAK xix

ABSTRACT xxi

CHAPTER 1: GENERAL INTRODUCTION

1.1 Research background 1

1.2 Problem statement 2

1.3 Objectives 4

CHAPTER 2: LITERATURE REVIEW

2.1 Polyhydroxyalkanoates (PHAs): The next generation bioplastics 5

2.1.1 PHAs in general 5

2.1.2 History and development of PHAs 10

2.1.3 Classification and properties of PHAs 15

2.1.4 Biosynthesis of PHAs 18

2.2 Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) 23

[P(3HB-co-4HB)]

2.2.1 Biosynthesis of P(3HB-co-4HB) copolymer 23

2.2.1(a) P(3HB-co-4HB) producers 24

2.2.1(a)(i) Cupriavidus necator 24

2.2.1(a)(ii) Delftia acidovorans 25

2.2.1(a)(iii) Alcaligenes latus 26

2.2.1(a)(iv) Other wild-type strains 27

2.2.1(a)(v) Recombinant Escherichia coli 29

2.2.1(b) Metabolic pathway of P(3HB-co-4HB) 30

2.2.2 Structure and properties of P(3HB-co-4HB) copolymer 32

iv

2.2.2(a) Molecular mass distribution 32

2.2.2(b) Physical and thermal properties 33

2.2.2(c) Biodegradability 37

2.2.2(d) Biocompatibility 39

2.3 Strategies in biotechnological production of P(3HB-co-4HB) 40

copolymer

2.3.1 P(3HB-co-4HB) producers from Malaysian environment 40

2.3.2 Conventional P(3HB-co-4HB) cultivation strategies 44

2.3.3 The mixed-substrate cultivation strategy 47

2.3.4 Bioprocess modeling and scale up bioprocess 49

2.3.4(a) Kinetics modeling 49

2.3.4(b) Oxygen transfer and scale up in 51

microbial bioprocess

2.4 Concluding remark 52

CHAPTER 3: MATERIALS AND METHOD

3.1 Chemicals and solvents 54

3.2 Sterilization 54

3.3 Centrifugation 54

3.4 Medium preparation 55

3.4.1 Nutrient rich (NR) broth 55

3.4.2 Nutrient agar (NA) 55

3.4.3 Mineral salts medium (MSM) 56

3.5 Bacterial strain and maintenance 56

3.5.1 Microorganism 56

3.5.2 Bacterial stock maintenance 57

3.5.3 Seed culture preparation 57

3.6 Single-stage biosynthesis of P(3HB-co-4HB) in 57

shake-flask

3.6.1 Effect of sole carbon precursor 58

3.6.2 Effect of substrate mixtures 58

v

3.7 Single-stage biosynthesis of P(3HB-co-4HB) in 59

controlled bioreactor system

3.7.1 Production of P(3HB-co-4HB) in 3 L fermentation 60

3.7.1(a) Effect of substrate mixing ratios 60

3.7.1(b) Effect of total carbon concentration 61

3.7.1(c) Kinetic modeling of P(3HB-co-4HB) 62

biosynthesis in single-stage mixed-substrate

cultivation

3.7.1(c)(i) Microbial growth 63

3.7.1(c)(ii) P(3HB-co-4HB) formation 63

3.7.1(c)(iii) Substrates utilization 64

3.7.1(c)(iv) Numerical calculations 65

3.7.2 Production of P(3HB-co-4HB) in 18 L and 70 L 65

fermentation

3.7.2(a) Determination of volumetric mass transfer 65

coefficient, KLa

3.7.2(b) Scale-up bioprocess of P(3HB-co-4HB) 66

3.8 Analytical procedures 67

3.8.1 Cell dry weight determination 67

3.8.2 Quantification of substrates in fermentation media 68

3.8.2(a) γ-butyrolactone, 1,4-butanediol and 68

1,6-hexanediol

3.8.2(b) Nitrogen 69

3.8.3 Quantification of PHA and monomer compositions 69

3.8.3(a) Preparation of methanolysis solution 69

3.8.3(b) Preparation of caprylic methyl ester (CME) 69

solution

3.8.3(c) Methanolysis 70

3.8.3(d) GC operation

3.8.3(e) Enumeration of PHA content and monomer 71

compositions

3.8.4 Extraction of P(3HB-co-4HB) copolymers 72

3.8.5 Polymer film casting 73

vi

3.9 Characterization of P(3HB-co-4HB) copolymers 73

3.9.1 Molecular weight 73

3.9.2 Mechanical properties 75

3.9.3 Thermal properties 76

3.9.4 X-ray crystallinity 76

3.9.5 Nuclear magnetic resonance (NMR) analysis 77

3.10 Microscopic observation 77

3.10.1 Phase contrast microscopy 77

3.10.2 Scanning electron microscopy (SEM) 77

3.10.3 Transmission electron microscopy (TEM) 78

3.10.3(a) Fixation of samples 78

3.10.3(b) Sectioning of resin blocks 79

3.11 Statistical analysis 80

CHAPTER 4: RESULTS AND DISCUSSION

4.1 Biosynthesis of P(3HB-co-4HB) in shake-flask scale 81

fermentation

4.1.1 Single substrate cultivation 81

4.1.2 Mixed-substrate cultivation 89

4.2 Manipulation of substrate mixing ratio in bioreactor system 95

4.2.1 Effect on bacterial growth and P(3HB-co-4HB) 95

accumulation

4.2.1(a) γ-butyrolactone and 1,4-butanediol 101

4.2.1(b) γ-butyrolactone and 1,6-hexanediol 106

4.2.1(c) 1,4-butanediol and 1,6-hexanediol 113

4.2.2 Effect on accumulation of 4HB monomer 118

4.3 Manipulation of total carbon concentration in bioreactor system 124

4.3.1 Effect on bacterial growth and P(3HB-co-4HB) 124

accumulation

4.3.2 Kinetic modeling of growth and P(3HB-co-4HB) 138

accumulation in single-stage mixed-substrate

cultivation system

vii

4.3.3 Effect on copolymer characteristics 146

4.4 Pilot scale production of P(3HB-co-4HB) 168

4.4.1 Effect of agitation and aeration on single-stage 168

fermentation of P(3HB-co-4HB)

4.4.2 Effect of agitation and aeration on copolymer’s 177

characteristics

4.4.3 Biosynthesis of P(3HB-co-4HB) in 70 L fermentation 180

CHAPTER 5: CONCLUSION

5.1 Summary 186

5.2 Recommendations for future work 189

REFERENCES 191

LIST OF PUBLICATIONS

APPENDICES

viii

LIST OF TABLES

Page

Table 2.1 General differences of SCL-PHAs and MCL-PHAs 17

Table 3.1 Compositions of NR broth 55

Table 3.2 Compositions of NA 55

Table 3.3 Compositions of MSM 56

Table 3.4 Compositions of trace elements dissolved up to 1 L 56

0.1 M hydrochloric acid (HCl)

Table 3.5 The combination strategies of the mixed-substrate 59

cultivation in shake-flask scale fermentation

Table 3.6 The combination strategies of the mixed-substrate 61

cultivation in bioreactor scale fermentation

Table 3.7 Variation of total carbon concentrations on selected 62

substrate mixtures

Table 3.8 Variation of agitation in 18 L fermentation 67

Table 3.9 Variation of aeration in 18 L fermentation 67

Table 3.10 Parameters for carbon leftover quantification in GC 68

Table 3.11 Parameters for PHA quantification in GC ` 71

Table 3.12 Parameters for molecular weight determination in GPC 74

Table 3.13 Samples washing solution and the time required for 78

SEM samples preparation

Table 3.14 Samples washing solution and the time required for 79

TEM samples preparation

Table 4.1 Effect of different concentration of γ-butyrolactone on 82

P(3HB-co-4HB) production

Table 4.2 Effect of different concentration of 1,4-butanediol on 83


Table 4.3 Effect of different concentration of 1,6-hexanediol on 84


Table 4.4 Production of copolymer P(3HB-co-4HB) using 90

combination of oleic acid with 4HB precursors

ix

Table 4.5 Production of copolymer P(3HB-co-4HB) using 91

combination among 4HB precursors

Table 4.6 The final bacterial growth and P(3HB-co-4HB) 96

accumulation at various substrate mixing ratios

Table 4.7 The final bacterial growth and P(3HB-co-4HB) 125

accumulation at various total carbon concentration

Table 4.8 Kinetics modelling for the bacterial growth, 143

P(3HB-co-4HB) accumulation and substrates utilization

for Lg+ H1,4, Lg+ H1,6 and H1,4+L1,6 at various

total carbon concentrations

Table 4.9 Molecular weight and thermal characterization 148

of P(3HB-co-4HB) synthesized using various

substrate mixtures

Table 4.10 Mechanical properties and crystallinity of 155

P(3HB-co-4HB) synthesized using various

substrate mixtures

Table 4.11 Mechanical properties of synthetic thermoplastics 162

Table 4.12 Comparison of P(3HB-co-4HB) synthesized with 162

various biodegradable

Table 4.13 Randomness of copolymer P(3HB-co-4HB) produced 163

Table 4.14 Effect of agitation and aeration on bacterial growth 169

and P(3HB-co-4HB) accumulation

Table 4.15 Effect of agitation and aeration on P(3HB-co-4HB) 178

copolymer’s characteristics

Table 4.16 Comparison of the P(3HB-co-4HB) production 181

and copolymer’s characteristics from various

fermentation scales

Table 4.17 Randomness of copolymers produced at various 185

fermentation scales.

x

LIST OF FIGURES

Page

Figure 2.1 General structure of poly(3-hydroxyalkanoates) in 9

which R refers to the length of side chain and n

refers to size of alkyl group. PHAs are made up

of carbon, hydrogen and oxygen atoms

Figure 2.2 The major pipelines in upstream and downstream 14

processing of PHAs

Figure 2.3 Metabolic pathway of P(3HB) synthesis 20

Figure 2.4 Metabolic pathways of mcl-PHAs biosynthesis 22

Figure 2.5 Differences between two-stage and single-stage 45

fermentation strategies

Figure 4.1 Effect of sole carbon precursors and concentrations 87

on the bacterial growth and copolymer accumulation

Figure 4.2 Transmission electron micrograph of Cupriavidus sp. 100

USMAA1020 with various PHA contents:

(a) 25 wt% from the substrates mixture of Hγ + L1,6;

(b) 55 wt% from the substrates mixture of Lγ + H1,4;

(c) 73 wt% from the substrates mixture of H1,4 + L1,6;

with 16,000x magnification. Bars represent 500 nm

Figure 4.3 Effect of γ-butyrolactone and 1,4-butanediol on 103

(a) residual biomass, (b) specific growth rate,

(c) PHA content, (d) PHA concentration,

(e) 4HB monomer composition and

(f) 4HB monomer concentration

Figure 4.4 Effect of γ-butyrolactone and 1,6-hexanediol on 108





Figure 4.5 Effect of 1,4-butanediol and 1,6-hexanediol on 114





Figure 4.6 Schematic pathway of P(3HB-co-4HB) formation from 122

γ-butyrolactone, 1,4-butanediol and 1,6-hexanediol

xi

Figure 4.7 Effect of total carbon concentration (0.42 wt% C, 127

0.56 wt% C and 0.69 wt% C) of γ-butyrolactone

and 1,4-butanediol on (a) cell dry weight, (b) residual

biomass, (c) PHA accumulation, (d) 4HB monomer

accumulation, (e) substrates leftover, (f) nitrogen

leftover of Cupriavidus sp. USMAA1020


0.56 wt% C and 0.69 wt% C) of γ-butyrolactone

and 1,6-hexanediol on (a) cell dry weight, (b) residual





0.56 wt% C and 0.69 wt% C) of 1,4-butanediol

and 1,6-hexanediol on (a) cell dry weight, (b) residual




Figure 4.10 Simulations of kinetic models and experimental results 140

for growth, P(3HB-co-4HB) production and substrates

leftover by Cupriavidus sp. USMAA1020 when the

medium was supplemented with Lγ + H1,4 at the total

carbon concentrations of 0.42 wt% C, 0.56 wt% C

and 0.69 wt% C. The model predictions and

experimental data are illustrated with solid lines and

symbols respectively




medium was supplemented with Lγ + H1,6 at the total








medium was supplemented with H1,4 + L1,6 at the total



xii



Figure 4.13 DSC thermograms for the copolymers produced 150

from various substrate mixtures at total carbon

concentration of 0.42 wt%C, 0.56 wt% C and

0.69 wt%C

Figure 4.14 The stress-strain curves of P(3HB-co-4HB) 156

copolymers produced from (a) 0.42 wt%C,

(b) 0.56 wt%C and (c) 0.69 wt%C γ + 1,4;

(d) 0.42 wt%C, (e) 0.56 wt%C and

(f) 0.69 wt%C γ + 1,6, (g) 0.42 wt%C,

(h) 0.56 wt%C and (i) 0.69 wt%C 1,4 + 1,6.

Figure 4.15 Crystallinity of (a-c) P(3HB-co-4HB) copolymers 158

with 27 mol%, 26 mol% and 22 mol% produced

from γ + 1,4, (d-f) P(3HB-co-4HB) copolymers with

30 mol%, 39 mol% and 51 mol% 4HB produced from

γ + 1,6, (g-i) P(3HB-co-4HB) copolymers with 32 mol%,

31 mol% and 41 mol% produced from 1,4 + 1,6.

Figure 4.16 The elastomeric P(3HB-co-4HB) bound back 161

almost to its original shape after being stretched

Figure 4.17 Comparison of expanded spectra of 3HB and 4HB 167

monomer unit at the respective carbonyl group

(C=O) of copolymer (a) P(3HB-co-39 mol% 4HB) from

0.56 wt% C γ + 1,6, (b) P(3HB-co-51 mol% 4HB) from

0.69 wt% C γ + 1,6, and (c) P(3HB-co-31 mol% 4HB)

from 0.56 wt% C 1,4 + 1,6 using (i) conventional

13C NMR and inverse gated decoupling (zgig)

13C qNMR analysis.

Figure 4.18 Effect of agitation on (a) residual cell weight; 171

(b) PHA accumulation and (c) 4HB monomer

accumulation of Cupriavidus sp. USMAA1020 in 18 L

fermentation using 0.56 wt% C H1,4+L1,6

Figure 4.19 Effect of aeration on (a) residual cell weight; 174

(b) PHA accumulation and (c) 4HB monomer

accumulation of Cupriavidus sp. USMAA1020 in 18 L

fermentation using 0.56 wt% C H1,4+L1,6

Figure 4.20 The growth of Cupriavidus sp. USMAA1020 and 183

P(3HB-co-4HB) copolymer accumulation in 70 L

fermentation.

xiii

Figure 4.21 Expanded 13C qNMR spectra of P(3HB-co-4HB) 184

copolymers produced from various fermentation scales

using 0.56 wt% C H1,4+L1,6: (a) 3 L, (b) 18 L and (c) 70 L.

The spectra represent the dyad sequence distribution of

3HB and 4HB monomer units at respective carbonyl

group (C=O).

xiv

LIST OF SYMBOLS AND ABBREVIATIONS

α Growth associated term

β Non-growth associated term

e Exponent

µmax Maximum specific growth rate

1H-NMR Proton-Nuclear magnetic resonance analysis

13C-NMR Carbon- Nuclear magnetic resonance analysis

ΔHm Enthalpy of fusion

3HB 3-hydroxybutyrate

3HV 3-hydroxyvalerate

3HHp 3-hydroxyheptanoate

3HHx 3-hydroxyhexanoate

3HO 3-hydroxyoctanoate

4HB 4-hydroxybutyrate

Aa Area under amorphous hump

Ac Area under crystalline peak

ANOVA Analysis of variance

ASTM American Society for Testing and Materials

ATCC American Type Culture Collection

C Carbon atom

C/N Carbon-to-nitrogen ratio

CoA Coenzyme A

C=O Carbonyl

CAM Chick chorioallantoic membrane

xv

CDCl3 Deuterated chloroform

CaCl2·2H2O Calcium chloride dehydrate

CuCl2·2H2O Copper (II) chloride dihydrate

CME Caprylic methyl ester

CoSO4·7H2O Cobalt (II) sulphate heptahydrate

D Randomness

Di Dispersity

DO Dissolved oxygen

FeSO4·7H2O Iron (II) sulphate heptahydrate

g Gravity

GC Gas chromatography

GC-MS Gas chromatography- mass spectroscopy

HA(s) Hydroxyalkanoate(s)

HMDS Hexamethyldisilazane

HPLC High performance liquid chromatography

ICI Imperial Chemical Industries

kDa Kilo Dalton

K2HPO4 Dipotassium hydrogen phosphate

KH2PO4 Potassium dihydrogen phosphate

KLa Volumetric mass transfer coefficient

LCL Long chain length

ms Maintenance coefficient

mol% Mol percentage

Mw Weight-average molecular weight

Mn Number-average molecular weight

MCL Medium chain length

xvi

MgSO4·7H2O Magnesium sulphate heptahydrate

MnCl2·4H2O Manganese (II) chloride tetrahydrate

MSM Mineral salt medium

NA Nutrient agar

NADH Nicotinamide adenine dinucleotide

(NH4)2SO4 Ammonium sulphate

N.D. Not detected

NMR Nuclear magnetic resonance

NR Nutrient rich

OD Optical density

PHA(s) Polyhydroxyalkanoate(s)

PhaA β-kethiolase

PhaB Acetoacetyl-CoA reductase

PhaC PHA synthase

PhaG (R)-3-hydroxyacyl-ACP-CoA-transferase

PhaP Phasin

PhaR Regulator protein for phasing expression

PhaZ PHA depolymerase

PLA Polylactic acid

P(3HB) P(3-hydroxybutyrate)

P(4HB) Poly(4-hydroxybutyrate)

P(3HB-co-3HV) Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

P(3HB-co-3HHx) Poly(3-hydroxybutyrate-co-3-

hydroxyhexanoate)

P(3HB-co-4HB) Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

P(3HB-co-4HV) Poly(3-hydroxybutyrate-co-4-hydroxyvalerate)

P(3HB-co-3HV-co-3HHx) Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-

co-3-hydroxyhexanoate)

xvii

P(3HB-co-3HV-co-4HB) Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-

4-hydroxybutyrate)

P(3HHx-co-3HO-co-3HD) Poly(3-hydroxyhexanoate-co-3-

hydroxyoctanoate-co-3-hydroxydecanoate)

P(3HHx-co-3HO-co-3HD-co- Poly(3-hydroxyhexanoate-co-3-

3HDD) hydroxyoctanoate-co-3-hydroxydecanoate-co-

3-hydroxydodecanoate)

P Product

P value Probability value

PDI Polydispersity index

PTFE Polytetrafluoroethylene

rpm Rotations per minute

s Substrate

SCL Short chain length

SSADH Succinate semialdehyde dehydrogenase

SEM Scanning electron microscope

sp. Species

spp. Several species

TEM Transmission electron microscope

TCA Tricarboxylic acid

TMS Tetramethylsilane

Tc Crystallization temperature

Tg Glass transition temperature

Tm Melting temperature

UV Ultraviolet

vvm Gas volume flow per unit of liquid volume per

minute (volume per volume per minute)

v/v Volume per volume

x Biomass

xviii

xc Crystallinity

xm Biomass at t = 0

xm Maximum biomass

Xc Degree of crystallinity

w/v Weight per volume

wt% Weight percentage

wt% C Weight percentage of carbon

Yx/s Yield coefficient of biomass over substrate

Yp/s Yield coefficient of product over substrate

Yp/x Yield coefficient of product over biomass

ZnSO4·7H2O Zinc sulphate heptahydrate

xix

BIOSINTESIS SATU PERINGKAT POLI(3-HIDROKSIBUTIRAT-ko-4-

HIDROKSIBUTIRAT) MENGGUNAKAN Cupriavidus sp. USMAA1020

MELALUI STRATEGI PENGKULTURAN SUBSTRAT CAMPURAN

ABSTRAK

Kopolimer poli(3-hidroksibutirat-ko-4-hidroksibutirat) [P(3HB-ko-4HB)]

merupakan polihidroksialkanoat (PHA) yang penting dalam bidang biofarmaseutikal

dan perubatan. Produktiviti kopolimer dengan komposisi 4HB yang tinggi sering

dicapai melalui pengkulturan dua-peringkat tetapi ini menyukarkan dengan

meningkatkan masa dan usaha pengendalian, dan risiko kontaminasi dalam proses

penghasilan berskala besar. Kajian ini bertujuan untuk membangunkan strategi

fermentasi satu-peringkat yang berkesan menggunakan strategi pengkulturan substrat

campuran sebagai pengganti fermentasi dua-peringkat. Strategi ini menunjukkan

kesan sinergi substrat campuran yang ketara terhadap pengumpulan P(3HB-ko-4HB)

secara satu-peringkat dengan peningkatan kandungan dan kepekatan PHA sebanyak

74% (b/b) dan 6.9 g/L masing-masing; dan komposisi monomer 4HB dari 7 mol% ke

70 mol%. Ianya adalah selaras dengan eksperimen menggunakan karbon tunggal

yang menunjukkan penghasilan PHA tertinggi sebanyak 57% (b/b), 3.1 g/L dan 48

mol% 4HB. Manipulasi substrat campuran dan nisbah di dalam bioreaktor

menunjukkan bahawa kebanyakan proses fermentasi telah menghasilkan kandungan

PHA yang melebihi 50% (b/b), dengan satu siri kopolimer yang mengandungi

komposisi monomer 4HB yang pelbagai dari 12 mol% ke 55 mol%. Penghasilan

PHA yang tinggi sebanyak 73% (b/b) telah diperolehi, dan seterusnya

menyumbangkan kepekatan dan hasil PHA yang tinggi sebanyak 8.6 g/L dan 2.7 g/g.

xx

Ciri-ciri kopolimer tersebut telah dipengaruhi oleh jenis substrat dan strategi

pencampuran. Bioproses satu-peringkat yang dijalankan menepati dengan baik model

logistik dan Luedeking-Piret (dan yang diubahsuai), dibuktikan dengan nilai R2 yang

tinggi dari 0.9153 ke 0.9957. Penggunaan kedua-dua substrat dan penghasilan

P(3HB-ko-4HB) adalah pada kadar yang tinggi pada fasa pertumbuhan eksponen

bakteria, dicirikan oleh nilai pemalar pertumbuhan-berkait penggunaan substrat (γ)

dan pengumpulan P(3HB-ko-4HB) (α) yang tinggi, masing-masing dari 0.0690 g/g

ke 0.2863 g/g dan 0.0660 g/g ke 0.1886 g/g. Hal ini adalah selari dengan nilai

pemalar pertumbuhan tak-berkait, δ dan β yang menunjukkan kadar penggunaan

substrat dan penghasilan kopolimer yang rendah, masing-masing dari 3.9 × 10-5 g/g/h

ke 0.0011 g/g/h dan 0.0092 g/g ke 0.0459 g/g pada fasa pertumbuhan pegun.

Kopolimer P(3HB-ko-30 mol% 4HB) yang mempunyai berat molekul, 927 kDa dan

pemanjangan-sehingga-putus tinggi dan 1637% telah dibiosintesiskan daripada

0.56% (b/b C) 1,4-butanadiol dan 1,6-hexanadiol dengan nisbah 3:1. Peningkatan

kadar goncangan dari 150 rpm ke 250 rpm telah menunjukkan peningkatan kadar

penghasilan P(3HB-ko-4HB) yang efisien masing-masing dari 28 % (b/b) ke 63%

(b/b) dan 3.1 g/L ke 6.5 g/L, yang meminimumkan penggunaan substrat terhadap

pertumbuhan bakteria dari 8.0 g/L ke 3.8 g/L. Kepelbagaian kadar pengudaraan dari

0.5 ke 1.0 vvm mengekalkan residu biojisim di antara 4.0 g/L dan 4.8 g/L tetapi

diiringi dengan peningkatan kepekatan PHA dari 4.8 g/L ke 7.2 g/L. Pengkulturan

substrat campuran telah terbukti sebagai strategi yang boleh meningkatkan

produktiviti kopolimer, komposisi monomer 4HB dan sifat kopolimer dalam

fermentasi secara satu-peringkat.

xxi

SINGLE-STAGE BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE-co-4-

HYDROXYBUTYRATE) USING Cupriavidus sp. USMAA1020 VIA MIXED-

SUBSTRATE CULTIVATION STRATEGY

ABSTRACT

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer

is polyhydroxyalkanoate (PHA) with biopharmaceutical and medical significance.

Copolymer productivity with high 4HB monomer composition is usually achieved

via two-stage cultivation but this reduces the ease of handling by increasing the time,

effort and risk of contamination in large-scale production. This study aims to by-pass

the two-stage fermentation by developing an efficient single-stage fermentation

strategy using mixed-substrate cultivation. This strategy showed significant

synergistic effect towards the single-stage production of P(3HB-co-4HB) with an

enhanced PHA content and concentration up to 74 wt% and 6.9 g/L respectively; and

4HB monomer composition from 7 mol% to 70 mol%. This is in relative to

experimentation using sole carbon sources which recorded the highest PHA

accumulation of 57 wt%, 3.1 g/L and 48 mol% 4HB. Manipulation of the substrate

mixture and its mixing ratios in bioreactor revealed that most of the fermentations

resulted in PHA content of more than 50 wt%, with a series of copolymers of 4HB

monomer compositions ranged from 12 mol% to 55 mol%. A remarkably high PHA

accumulation of 73 wt% was obtained, contributing to high PHA concentration and

yield of 8.6 g/L and 2.7 g/g respectively. The copolymer characteristics were

predominantly affected by the nature of substrates and the mixing strategies. The

single-stage fermentation fit well to the logistic and Luedeking-Piret (and modified)

models, evidenced by high adjusted R2 values of 0.9153 to 0.9957. Both substrates

xxii

utilization and P(3HB-co-4HB) accumulation were at higher rate during bacterial

exponential growth phase, characterized by higher values of growth-associated

substrate utilization (γ) and P(3HB-co-4HB) accumulation (α) constants of 0.0690

g/g to 0.2863 g/g and 0.0660 g/g to 0.1886 g/g respectively. This is in relative to

non-growth associated constants, δ and β which demonstrated lower substrates

utilization and copolymer accumulation rate during stationary phase respectively at

3.9 × 10-5 g/g/h to 0.0011 g/g/h and 0.0092 g/g to 0.0459 g/g. A P(3HB-co-30 mol%

4HB) copolymer with high molecular weight and elongation at break values of 927

kDa and 1637% was biosynthesized from 0.56 wt% C 1,4-butanediol and 1,6-

hexanediol at ratio of 3:1. An increase of agitation from 150 rpm to 250 rpm has

resulted in increase efficiency of P(3HB-co-4HB) accumulation from 28 wt% to 63

wt% and 3.1 g/L to 6.5 g/L respectively which concurrently minimized the utilization

of substrates towards bacterial growth from 8.0 g/L to 3.8 g/L. Variation of aeration

from 0.5 vvm to 1.0 vvm kept the residual biomass constant in between 4.0 g/L and

4.8 g/L but accompanied with an increase of PHA concentration from 4.8 g/L to 7.2

g/L. The mixed-substrate cultivation was proven to be promising strategy to enhance

copolymer yield, 4HB accumulation and characteristics in single-stage fermentation.

1

CHAPTER 1: INTRODUCTION

1.1 Research background

Polyhydroxyalkanoates (PHAs) are group of biopolymers made up of diverse

range of monomers. To date, over 150 monomer constituents have been identified as

building blocks of PHAs (Gao et al., 2011; Urtuvia et al., 2014; Jia et al., 2016). Of

all PHAs, poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)]

copolymer is known to be suitable absorbable biomaterial in medical and

pharmaceutical applications. It can be specifically applied for wound healing,

orthopedics, therapeutics, drug delivery, tissue engineering and etc (Koller et al.,

2007; Chee et al., 2008; Wu et al., 2009; Brigham and Sinskey, 2012; Luef et al.,

2015; Phong et al., 2016).

There are few reasons directing the usage of the copolymer as high value

medical product. Unlike other PHAs, this copolymer was found not only degraded by

depolymerases, but also capable of hydrolyzation by lipases and esterases from both

prokaryotes and eukaryotes (Doi et al., 1992; Saito et al., 1996; Li et al., 2010; Hsieh

et al., 2009; Vigneswari et al., 2016). This is due to the absence of alkyl side chain as

pendant group on 4HB monomers being attached to the copolymer chain (Mukai et

al., 1993; Saito et al., 1996). The introduction of 4HB monomer results in

copolymers with higher in vivo degradation rate than other PHAs (Loo and Sudesh,

2007; Li et al., 2010). Besides, the degradation of P(3HB-co-4HB) releases 3-

hydroxybutyric acid and 4-hydroxybutyric acid which are natural metabolites

presence in human bloodstream (Bessman and Fishbein, 1964; Lee, 1996a; Martin

and Williams, 2003). This enhances the biocompatibility as the copolymer has the

ability to tolerate inflammatory responses with tissue, without imposing detrimental

2

side effect on human body. In term of the material properties, the copolymer exhibits

wide variety of mechanical strengths from highly crystalline plastic to elastomeric

rubber-like material, upon combining both 3HB and 4HB monomer constituents at

varying molar fractions (Doi et al., 1990; Saito et al., 1996; Loo and Sudesh, 2007;

Cesário et al., 2014). This improves the potential of biomaterial applications since

the resulting copolymers can be tailored to meet specific needs. P(3HB-co-4HB)

copolymer is considered to be one of the most valuable biopolymer among the vast

number of PHAs being synthesized by numerous microorganisms (Amirul et al.,

2008; Park and Kim, 2011).

1.2 Problem statement

P(3HB-co-4HB) is produced via fermentation process rather than chemical

synthesis (Martin and Williams, 2003). To date, most of the fermentations targeting

on this copolymer uses two- or three-stage cultivation in order to obtain high PHA

productivity and copolymers with high 4HB monomer composition (Doi et al., 1988;

Kunioka et al., 1989a; Doi et al., 1990; Nakamura et al., 1992; Saito et al., 1996;

Choi et al., 1999; Mitomo et al., 2001; Kim et al., 2005; Lee et al., 2007; Amirul et

al., 2008; Hsieh et al., 2009; Vigneswari et al., 2009 Rao et al., 2010a). This includes

the production of poly(4-hydroxybutyrate) [P(4HB)] homopolymer as well (Saito

and Doi, 1994; Saito et al., 1996; Choi et al., 1999). Obtaining copolymers with

higher 4HB monomer composition is desirable as it is generally concluded that this

would enhance the copolymer’s application as biomaterial in medical and bio-

pharmaceutical (Martin and Williams, 2003). However, multi-stages fermentation

strategy is not feasible when it comes to industrial scale production. This bioprocess

requires a minimum of two fermentation vessels which involves the use of nutrient-

rich medium for bacterial growth phase, followed by nitrogen-free medium for PHA

3

accumulation phase in separate vessels. This reduces the ease of handling, prone to

the risk of contamination during inocula transfer and indirectly increases the

operational cost and effort (Rahayu et al., 2008). Production of P(3HB-co-4HB) via

single-stage cultivation is less frequently employed and usually returned with either

low copolymer productivity and/or low 4HB monomer composition. For example,

Park and Kim (2011) were able to produce high biomass of 10 g/L to 20 g/L but

resulted in accumulation of low 4HB monomer composition of 6 mol% to 10 mol%.

Kang et al. (1995) were able to produce P(3HB-co-4HB) with high 4HB monomer

composition up to 83 mol% but with low PHA concentration of 0.4 g/L. In spite of

this, single-stage cultivation still offers an advantage in industrial-wise for the

production of P(3HB-co-4HB).

To date, the production of this copolymer has not been extensively explored

in bioreactor scale through single-stage fermentation. This is also true even for two-

stage fermentation process which normally resulted in production of homopolymer

P(4HB) or copolymer P(3HB-co-4HB) with high 4HB monomer composition. Most

of the two-stage fermentations were carried out in shake-flask fermentation (Renner

et al., 1996; Saito et al., 1996; Choi et al., 1999; Mitomo et al., 2001; Lee et al.,

2004; Kim et al., 2005; Chai et al., 2009; Rao et al., 2010b; Vigneswari et al., 2010).

Several recent studies have reported the copolymer production through single-stage

fermentation in bioreactor but these did not return with copolymer of higher

productivity and higher 4HB monomer composition (Park and Kim, 2011;

Cavalheiro et al., 2012; Cesário et al., 2014). The copolymers produced have 4HB

monomer compositions of less than 15 mol%.

Overall, there are some limitations on the upstream biosynthesis of the

copolymer. This will eventually reduce the feasibility of the copolymers to be

4

applied in healthcare application. In light of this, investigation and search for an

efficient approach that will optimally produce the copolymer is significantly

important.

1.3 Objectives

In the present study, the P(3HB-co-4HB) copolymer was biosynthesized by

Cupriavidus sp. USMAA1020 isolated from Malaysian environment. The novelty of

the work comes from the idea of relating the mixed-substrate cultivation strategy on

the single-stage production of the P(3HB-co-4HB) copolymer. To the best of my

knowledge, there are limited publications which discussed the effect of the strategy

on P(3HB-co-4HB) production in details. It is hypothesized that substrate mixture

strategy would overcome the limitation in single-stage cultivation, leading to the

development of efficient bioprocess strategy for the copolymer production.

The objectives of this study are:

1. To evaluate the applicability of mixed-substrate cultivation in improving single-

stage production of P(3HB-co-4HB) through shake-flask scale fermentation.

2. To produce P(3HB-co-4HB) copolymers from single-stage mixed-substrate

cultivation in controlled bioreactor system.

3. To study the production kinetics and characteristics of P(3HB-co-4HB)

biosynthesized by Cupriavidus sp. USMAA1020 in single-stage mixed-substrate

cultivation system.

4. To investigate the parameters affecting the upscaling process of P(3HB-co-4HB)

from lab to pilot scale fermentation.

5

CHAPTER 2: LITERATURE REVIEW

2.1 Polyhydroxyalkanoates (PHAs): The next generation bioplastics

2.1.1 PHAs in general

Polyhydroxyalkanoates (PHAs) are a group of natural biopolymer consisting

of a repeated chain of hydroxyalkanoates (HAs) monomer. These biopolymers are

synthesized by a wide variety of microorganisms under stress conditions whereby the

surrounding environments are limited of nutrients such as nitrogen, magnesium,

phosphorus or oxygen but sufficient with carbon sources (Anderson and Dawes,

1990; Liu et al., 2016).

PHAs are identified as one of bacterial storage components analogous to

starch and glycogen (Dawes and Senior, 1973; Olukoshi and Packter, 1994; Sudesh

et al., 2000). PHAs are lipoidic and belong to one of the four principal classes of the

storage polymers namely lipids, carbohydrates, polyphosphates and nitrogen reserve

compounds found in microorganisms (Dawes, 1985). Under the restricted microbial

growth conditions, the carbon sources are converted to PHAs, which serves as

intracellular carbon and energy storage compounds. PHAs exhibit high degree of

polymerization by the action of PHA synthase, with the resulting molecular weight

up to several million Daltons (Anderson and Dawes, 1990; Valappil et al., 2007).

PHAs exist in the form of discrete inclusion bodies, typically 0.2 µm to 0.5

µm in diameter; and localized in a mobile amorphous state within the cytoplasm of

the cells (Dawes and Senior, 1973; Lee, 1996a; Sudesh et al., 2000). These granules

are highly refractive and can be clearly visualized under phase contrast and electron

microscopic observation (Dawes and Senior, 1973; Lee, 1996a; Sudesh et al., 2000).

PHAs are water-insoluble. This makes PHA an excellent energy reserve material

6

since it does not contribute to any significant increase in osmotic pressure within the

bacterial cytoplasm, even in a high quantity of PHAs (Loo and Sudesh, 2007). As the

physiological fitness of the cells is not affected, this would prevent the leakage of

valuable compounds out of the cells which in turn securing the stored nutrients at

low maintenance cost (Peters and Rehm, 2005; Rehm, 2006; Verlinden, 2007).

PHAs are synthesized by various bacterial species from eubacteria and

archeabacteria with a total of more than 90 generas (Zinn et al., 2001; Kessler et al.,

2001; Rehm, 2006). This includes numerous Gram-positive and Gram-negative

bacteria in which the former is prominently represented by Bacillus sp., Streptomyces

sp., Rhodococcus sp., Clostridium sp., Staphylococcus sp., Corynebacterium sp., and

Nocardia sp.; whereas the latter is notably denoted to Cupriavidus sp., Pseudomonas

sp., Methylobacterium sp., Azotobacter sp., Burkholderia sp. and recombinant

Escherichia coli (Lee, 1996a; Valappil et al., 2007; Liu et al., 2016).

Among all these, the Gram-negative Cupriavidus necator (formerly Ralstonia

eutropha, Alcaligenes eutrophus or Waustersia eutropha) is the representative of

PHA producers and extensively studied in PHA research (Jendrossek, 2009;

Tanadchangsaeng and Yu, 2012). This is because the bacterium, of strictly

respiratory and facultative chemolithoautotrophic is able to utilize a diverse range of

carbon sources from simple, defined to complex and inexpensive for growth and

PHA biosynthesis (Kim et al., 1994; Sugimoto et al., 1999; Valappil et al., 2007;

Ashby et al., 2011). Besides, the bacterium can accumulate high quantities of PHA

up to 90 wt% of the biomass under appropriate nutrient depleted conditions

(Jendrossek and Handrick, 2002; Khanna and Srivastava, 2005; Chen, 2009;

Tanadchangsaeng and Yu, 2012).

7

At present, PHA producing microorganisms are found in either natural or

engineered ecosystems such as in soil, marine, wastewater treatment plant, salt marsh

and activated sludge process (Liu et al., 2016). The production of PHAs is not

feasible via chemical synthesis but through in vivo accumulation by microorganism

itself as processing factory (Valappil et al., 2007; Wang et al., 2014). Therefore,

industrial scale production of successfully commercialized PHAs has usually been

performed using enzyme and bioprocess engineering approach (Liu et al., 2016).

Among the various biodegradable polymers, PHAs are excellent substitutes

for petroleum-based synthetic plastics because they exhibit plastic-like feature in

term of the physical and thermal properties. They are water resistance, oxygen

impermeable and piezoelectric thermoplastics and/or elastomers (Valappil et al.,

2007; Keshavarz and Roy, 2010). Unlike conventional plastics, PHAs are known and

recognized to be the only waterproof thermoplastics that are completely

biodegradable in terrestrial and aquatic ecosystems by microorganisms (Philip and

Roy, 2007). These polymers are degraded into harmless by-products, releasing

carbon dioxide and water under aerobic conditions; methane and water under

anaerobic conditions (Jendrossek and Handrick, 2002; Lim et al., 2005; Sridewi et

al., 2006; Chanprateep, 2010). Without releasing any toxic waste, this resolves the

issue of toxicity caused by the accumulation of non-degradable plastic waste.

In addition, PHAs are completely being biosynthesized form renewable

resources. This resolves the issue of over dependence on synthetic plastics which

uses fossil fuels that are facing depletion as raw materials (Zinn et al., 2001;

Steinbüchel, 2005; Chanprateep, 2010). PHAs have also caught attention due to their

biocompatibility. They are immunologically inert which is promising in fabricating

high-value medically related products.

8

PHAs are group of biopolymers made up of a diverse range of monomers.

Depending on the substrate specificity of PHA synthase of the particular strain,

various homopolymers, copolymers, terpolymers, blocks and blends PHAs could be

synthesized. To date, there are more than 150 monomer constituents found to be

produced by more than 300 microorganisms. The structural diversity of the

monomers and compositions has resulted in PHAs with a wide range of physical and

thermal characteristics. This has opened up to various opportunities for exploitation

as there are many possibilities yet to be discovered in various applications. Figure 2.1

shows the general chemical structure of various PHAs.

Owing to the renewable, eco-friendly and the diversification of the material

properties, PHAs are therefore can be applied into various sectors from disposable

goods packaging, agriculture, aquacultural, to medical and biopharmaceutical

industries (Chen and Wu, 2005; Tanadchangsaeng and Yu, 2012). PHAs are

envisioned to be one of the next generation materials which will return lots of

benefits to mankind and the world.

9

n = 1 R = hydrogen Poly(3-hydroxypropionate)

R = methyl Poly(3-hydroxybutyrate)

R = ethyl Poly(3-hydroxyvalerate)

R = propyl Poly(3-hydroxyhexanoate)

R = pentyl Poly(3-hydroxyoctanoate)

R = nonyl Poly(3-hydroxydodecanoate)

n = 2 R = hydrogen Poly(4-hydroxybutyrate)

R = methyl Poly(4-hydroxyvalerate)

n = 3 R = hydrogen Poly(5-hydroxyvalerate)

R = methyl Poly(5-hydroxyhexanoate)

n = 4 R = hexyl Poly(5-hydroxydodecanoate)

Figure 2.1: General structure of poly(3-hydroxyalkanoates) in which R refers to the

length of side chain and n refers to size of alkyl group. PHAs are made up of carbon,

hydrogen and oxygen atoms (Lee, 1996b)

O C (CH2)n C

R

H O 100-30000

10

2.1.2 History and development of PHAs

PHAs have gone through for almost a century since they have been

discovered (Keshavarz and Roy, 2010). Although the commercialization of PHAs

have still returned in limited success, tremendous efforts have been carried out which

expands the fundamentals of the microbial physiology in PHA biosyntheses, leading

to the established manipulation of the production through multi-level omics (Chen,

2009). According to Sudesh et al. (2000), the science and technology development of

PHAs is significantly marked by three stages: discovery of P(3HB), identification of

various hydroxyalkanoate monomers other than 3HB and manipulation of PHA

biosynthesis genes. Along this timeline, the development is also closely related to the

establishment and the improvement of PHA quantification method.

The presence of lucent granules in bacterial cytoplasm was first reported by

Beijerinck in 1888 (Amara and Moawad, 2011; Tanadchangsaeng, 2014). However,

the composition of PHA was only characterized at the mid of 1920s, by a French

microbiologist, Maurice Lemoigne who identified the inclusion bodies found in

Bacillus megaterium as poly(3-hydroxybutyrate) [P(3HB)] (Anderson and Dawes,

1990). By 1950s, accumulated studies from genus Bacillus suggested that P(3HB)

functions as intracellular carbon and energy reserve. Dawes and Senior (1973) later

on reviewed the physiological roles and concluded that the function of P(3HB) is

analogous to starch and glycogen which are well known bacterial storage materials.

Lemoigne was also the first who reported the analytical method for P(3HB).

This has been performed via long processes including extraction, purification,

saponification and distillation. These processes resulted in volatile and non-volatile

fractions which both contains the polymer respectively in different forms, trans-2-

11

butenoic acid (dehydrated 3-hydroxybutyric acid) and 3-hydroxybutyric acid. The

compounds were weighed and represented as accurate amount of P(3HB) found in

the bacterial cells.

Lemoigne’s method was accurate but requires large biomass quantity and the

whole process was time-consuming. To overcome this, various improvements have

been carried out such as using spectrophotomeric method by Williamson and

Wilkinson (1958), Slepecky and Law (1960) and Law and Slepecky (1961); glass

fibre disk assay method by Ward and Dawes (1973) which permits faster and

handling of smaller samples. Karr et al. (1983) and Hesselmann (1999) developed

the P(3HB) quantification using ion exchange high-performance liquid

chromatography (HPLC) and UV measurement.

The 3-hydroxybutyrate (3HB) was the only known hydroxyalkanoate (HA) to

be the building block of this polymer storage until 1970s. Wallen and Rohwedder

(1974) discovered the presence of HAs other than 3HB in the chloroform extracts of

activated sewage sludge. Analysis using nuclear magnetic resonance (NMR)

revealed the presence of longer hydroxy fatty acids meanwhile the subsequent

analysis by gas chromatography-mass spectroscopy (GC-MS) confirmed the

presence of 3-hydroxyvalerate (3HV) and 3-hydroxyhexanoate (3HHx). Findlay and

White (1983) detected that at least 11 HAs with 3HB and 3HV being major

constituents in the polymer extracted from marine sendiments whereas 3HB, 3-

hydroxyheptanoate (3HHp) and 3-hydroxyoctanoate (3HO) were found in the

polymer extracted from the monocultures of Bacillus megaterium. Odham et al.

(1986) reported the presence of 3HB, 3HHx, and 3HO from activated sewage sludge.

de smet et al. (1983) and Lageveen et al. (1988) significantly reported the

accumulation of 3HO and 3HHx without 3HB from Pseudomonas oleovorans, when

12

grown on n-octane. The PHA quantification method has also come to its major

advancement to this end. The method which consists of acidic based extraction,

hydrolysis and methylation developed by Braunegg et al. (1978) provides accurate

and more reproducible analytical measurement. The quantification method using gas

chromatography (GC) was applied and adapted widely by many authors.

The identification of other HA units marks a significant impact in PHA

research, development and commercialization. This is due to the fact that

homopolymer P(3HB) is brittle that limited the application as a substitute for

synthetic plastic. Therefore, incorporation of foreign HA units into the P(3HB)

polymer chain could enhance the overall material properties which extends the

material applications.

In early 1960s, PHA received its commercial attention by W.R. Grace & Co.

(U.S.A) which patented the fermentation process of P(3HB) and its fabrication into

absorbable prosthetic devices. However, the industrial scale exploitation of P(3HB)

are remained inactive. Following the petroleum crisis in 1970s, the demand of

searching alternatives for plastic industry has boosted. The Imperial Chemical

Industries, ICI from United Kingdom (currently known as Zeneca Bio Products) has

initiated the production of 70 wt% P(3HB) from Acaligenes latus. Besides, P(3HB-

co-3HV) copolymer was also produced and marketed under the trademark

BIOPOL® by the same company. Monsanto, a splitting of ICI has later acquired the

BIOPOL® and produces various range of P(3HB-co-3HV) copolymers in April

1996. After two years, Metabolix Inc. obtained license from Monsanto and launched

a company named Tepha Inc. which developed the medical devices using PHAs

(Braunegg et al., 1998; Philip and Roy, 2007). Biotechnologische

Forchungsgesellschaft is another company in Austria that has also developed the

13

pilot scale production of P(3HB) in 1990 (Hänggi, 1990; Hrabak, 1992; Kessler et

al., 2001). The process which used sucrose as sole carbon source would able to

accumulate the homopolymer up to 80% of cell dry weight. A biomass density of 60

g/L was achieved but the production process stopped in 1993 (Kessler et al., 2001).

The revolution of molecular biology approach in life science research has

encouraged the understanding of PHA biosynthesis both at genetic and enzymatic

level. By 1980s, genes that are responsible for PHA biosynthesis were successfully

cloned from Ralstonia eutropha into Escherichia coli. Ralstonia eutropha was

extensively studied as model for PHA biosynthesis and this revealed three important

enzymes responsible in producing P(3HB) from acetyl-CoA. Up to year 2003, there

was about 60 PHA synthase structural genes were cloned and sequenced from

various eubacteria (Steinbüchel and Lutke-Eversloh, 2003; Kadouri et al., 2005).

Generally, the development of PHAs can be categorized into upstream and

downstream processing in which the former involves the biosynthesis of PHAs and

the latter involves the characterization and application of PHAs as presented at

Figure 2.2. The upstream processing of PHAs includes the isolation of

microorganisms, screening and optimization, genetic modification and biosynthesis

at various fermentation scales; meanwhile the downstream processing of PHAs

covers the recovery and purification of the biopolymers, degradation, quantification,

characterization as well as the application of PHAs.

14

Figure 2.2: The major pipelines in upstream and downstream processing of PHAs

(Amirul et al., 2015)

15

2.1.3 Classification and properties of PHAs

PHAs can be categorized based on its monomeric units. PHAs with three

carbons (C3) to five carbons (C5) monomers are classified as short-chain-length

(SCL-). PHAs with the range from C6 to C14 are classified as medium-chain-length

(MCL).

SCL-PHAs are stiff, brittle and crystalline materials that contains methyl or

ethyl group as small side chains (Verlinden et al., 2007). Cupriavidus necator is one

of the bacteria that produces SCL-PHAs (Matsusaki et al., 1998). Homopolymer

P(3HB) is a typical example of SCL-PHA. It possesses high tensile strength that is

comparable to commodity plastic such as polypropylene, but with a low elongation at

break (Anderson and Dawes, 1990; Khanna and Srivastava, 2005). P(3HB) is rigid

and brittle with low elasticity. This is due to its perfect stereoregularity which

acquires detrimental aging process at ambient temperatures (de Koning and Lemstra,

1993). It is highly crystalline which consists of large spherulites and possesses post-

crystallization behaviours. As a result, formation of the irregular pores on the surface

limits the flexibility of amorphous polymer chain (Shishatskaya and Volova, 2004;

Wen et al., 2012; Hong et al., 2011). The homopolymer exhibits high melting

temperature at 170°C to 180°C. As a whole, this homopolymer is difficult to process

and therefore unfavourable to be applied for industrial use.

Even though P(3HB) is the most studied and well-characterized PHA, it

should be noted that its characteristics do not represent the overall material properties

of SCL-PHAs. By incorporating other hydroxyalkanoate units into the P(3HB)

macromolecular chain, this will result in enhanced material properties following the

disturbed and reduced crystal lattice arrangement of P(3HB) (Doi, 1995). This is

16

mainly accomplished by the addition of carbon precursors as co-substrate during the

upstream biosynthesis process, which subsequently copolymerizes 3-

hydroxybutyrate and other monomers. The PHAs with improved material properties

such as P(3HB-co-3HV), P(3HB-co-4HB), P(3HB-co-4HV), P(3HB-co-3HV-co-

4HB) are some of the known examples of SCL-PHAs.

The discovery of the first MCL-PHA was dated on 1983, in which a polyester

containing mainly of 3-hydroxyoctanoic acid from an axenic culture of Pseudomonas

oleovorans (de smet et al., 1983; Koller et al., 2013). It was later found out that most

of the bacteria from genus Pseudomonas (belong to rRNA-homology-group I) are

capable to synthesize MCL-PHA, with only a few exceptions (Steinbüchel and

Lutke-Eversloh, 2003). Typical MCL-PHAs includes P(3HHx-co-3HO-co-3HD) and

P(3HHx-co-3HO-co-3HD-co-3HDD). These polymers are generally elastomeric and

semi crystalline, which possesses low tensile strength and high elongation to break.

In term of thermal properties, MCL-PHAs exhibits lower glass transition temperature,

lower degree of crystallinity and broader melting range as compared to SCL-PHAs.

Apart from that, these polymers also possess lower molar mass due to a low degree

of polymerization (Koller et al., 2013). Table 2.1 shows general difference in

between SCL-PHAs and MCL-PHAs.

Since SCL-PHAs are stiff and brittle whereas MCL-PHAs are more

elastomeric and flexible, incorporation of both SCL- and MCL- monomers will result

in a SCL- MCL- copolymer which possesses the properties in between two states.

The resulting polymer has superior properties as compared to either SCL- or MCL-

PHAs. According to Chen (2009), SCL-MCL-PHAs are ideal in advancing the on-

going development in various applications since they exhibit flexible mechanical

properties. An attempt in producing this combination of copolymers started since

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1988 by Brandl et al. (1989) who studied the ability of Rhodospirillum rubrum, a

phototrophic bacterium to produce various kinds of PHAs. Among all the SCL-

MCL-PHAs produced, P(3HB-co-3HHx) is one of the successful polymer in this

category been produced up to an industrial scale (Chen, 2009). P(3HB-co-3HV-co-

3HHx) terpolymer is another SCL-MCL-PHA which has returned novel and

attractive properties to be employed in various application (Bhubalan et al., 2008).

Pocter & Gamble has also trademarked SCL-MCL-PHA copolymers as NodaxTM

which comprising of C4 and C6-C12 monomers (Bugnicour et al., 2014).

Table 2.1: General differences of SCL-PHAs and MCL-PHAs (Koller et al., 2013;

Amirul et al., 2015)

SCL-PHAs Properties MCL-PHAs

Around 0 °C Glass transition

temperature (Tg)

Around -40 °C

Up to 70% Degree of crystallinity (Xc) Not more than 40 %

Higher, up to 180 °C Melting point (Tm) Broad range of melting

temperature

Higher Molar mass Lower

Higher Polydispersity (PDI) Lower

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2.1.4 Biosynthesis of PHAs

Since the first discovered PHA, P(3HB) was found to play significant role as

carbon and energy source during imbalanced growth conditions, this has led to a

remarkable interest in the metabolic pathways and regulatory metabolism of the PHA

biosynthesis. The biosynthesis and utilization of PHAs are mediated by important

enzymes such as PHA synthases (PhaC) and PHA depolymerases (PhaZ). PHA

synthase is among the key enzyme that exclusively involved in PHA accumulation

since it polymerizes the β-hydroxyalkanoyl-CoAs into poly-β-hydroxyalkanoate

(Kadouri et al., 2005). PHA synthases are localized onto the surface of PHA granules

together with phasins (PhaP)/regulator (PhaR) system which also involves in

promoting PHA accumulation (York et al., 2002).

Bacterial PHA synthases can be classified into Class I, Class II, Class III and

Class IV PHA synthases in according to the subunit composition (either only PhaC,

or PhaC with PhaE) and the substrate specificity (resulting in either SCL-PHAs or

MCL-PHAs). Class I PHA synthases are known as SCL-PHA synthases, composed

one type of subunit and use C3-C5 monomers as substrates; the MCL-PHA synthases

are Class II synthases which also composed of one type of subunit but using C6-C14

as substrates; meanwhile Class III PHA synthases uses C3-C5 substrates and

composed of two different subunits. The three classes of the PHA synthases are

generally represented by Ralstonia eutropha (Cupriavidus spp.), Pseudomonas

aeruginosa (Pseudomonas spp.) and Allochromatium vinosum respectively

(Stainbuchel and Lutke-Eversloh, 2003; Kadouri et al., 2005; Philip and Roy, 2007;

Hyakutake et al., 2014; Ushimaru et al., 2014). Tsuge et al. (2015) highlighted the

recent investigation of newest classification of PHA systhases. The class IV

synthases composed of PhaC and PhaR subunit and are prevalent among

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microorganism from Bacillus genus such as Bacillus megatarium and Bacillus

cereus.

The biosynthesis of P(3HB) is the best known PHA biosynthetic pathway.

This SCL-PHA involves a three-step enzymatic reaction. In this pathway, β-

kethiolase (PhaA) catalyzes the formation of acetoacetyl-CoA from two acetyl-CoAs

via condensation reaction. This is followed by the action of acetoacetyl-CoA

reductase (PhaB) which reduces acetoacetyl-CoA to 3-hydroxybutyryl-CoA by

NADH. Finally, PHA synthase (PhaC) polymerizes the 3-hydroxybutyryl-CoA to

form P(3HB) via esterification reaction and release coenzyme-A. P(3HB)

biosynthesis is regulated by intracellular concentration of acetyl-CoA and 3-

kethiolase. During balanced growth conditions, acetyl-CoAs are channeled towards

tricarboxylic (TCA cycle). This releases a high amount of free coenzyme A which

inhibits the activity of 3-kethiolase. When the growth is limited, the level of NADH

and acetyl-CoA increases. Thus, the concentration of free coenzyme A reduced. This

promote the activity of 3-kethiolase which stimulates P(3HB) biosynthesis (Zinn et

al., 2001; Loo and Sudesh, 2007; Verlinden et al., 2007). Figure 2.3 shows the

metabolic pathway of P(3HB) biosynthesis.

The biosynthesis of SCL-PHAs other than P(3HB) occurs by feeding the

structurally related precursor substrates to the microorganism. The production of

these PHAs depends on the metabolism level required to convert the substrate

precursors into corresponding hydroxyacly-CoA thioesters, as well as the broad

substrate range of the PHA synthases (Steinbüchel and Valentin, 1995; Steinbüchel

and Fuchtenbusch, 1998). For example, P(3HB-co-3HV) copolymer can be produced

in most P(3HB) accumulating strain by feeding substrates such as pentanol,

propionic acid, and valeric acid with odd number of carbon atoms. These substrates

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Figure 2.3: Metabolic pathway of P(3HB) synthesis (Anderson and Dawes, 1990)

Acetyl-CoA + Acetyl-CoA

CoAS

Acetoacetyl-CoA

NADPH + H+

NADP+

(R)-3HB-CoA

P(3HB)

Step 1

3-ketothiolase

Step 2

Acetoacetyl-reductase

Step 3

PHA synthase

SINGLE-STAGE BIOSYNTHESIS OF POLY(3- HYDROXYBUTYRATE...

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