UNIVERSITI PUTRA MALAYSIA

PRODUCTION OF ORGANIC ACIDS FROM RESTAURANT WASTES AS SUBSTRATES FOR POLYHYDROXYBUTYRATE PRODUCTION BY

RECOMBINANT E. COLI

MAJD KHALID ESHTAYA

FK 2007 34

PRODUCTION OF ORGANIC ACIDS FROM RESTAURANT WASTES AS

SUBSTRATES FOR POLYHYDROXYBUTYRATE PRODUCTION BY RECOMBINANT E. COLI

By

MAJD KHALID ESHTAYA

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of Science

June 2007

DEDICATION

ALLAH YOU ARE MY SUPREME LOVE

ALHAMDULILLAH ….THANK YOU FOR EVERYTHING

TO MY BELOVED COUNTRY PALESTINE

MY BELOVED PARENTS, SISTERS AND BROTHERS

THANKS FOR YOUR DOA’, LOVE AND CARE

FRIENDS

THANKS FOR YOUR HELP AND SUPPORT

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

PRODUCTION OF ORGANIC ACIDS FROM RESTAURANT WASTES AS SUBSTRATES FOR POLYHYDROXYBUTYRATE PRODUCTION BY

RECOMBINANT E. COLI

By

MAJD ESHTAYA

June 2007

Chairman: Professor Mohd. Ali Hassan, PhD

Faculty: Engineering

In this study, restaurant waste was utilized as sole carbon source for the production of

polyhydroxybutyrate (PHB) by recombinant Escherichia coli. PHB production was

achieved via a two-stage process; organic acids production from fermented restaurant

waste followed by PHB production utilizing the organic acids. In the first stage, the

effect of initial pH, operating temperature and enzymatic pretreatment (hydrolysis using

α-amylase and glucoamylase) were investigated to get highest organic acids production

from restaurant wastes in anaerobic treatment. Anaerobic treatment was carried out in

500 mL Erlenmeyer flask using orbital shaking incubator. Three different temperatures

were chosen; 30°C, 37°C and uncontrolled (room temperature). The initial pH of

fermentation was also varied; adjusted at 7 and without adjustment. The organic acids

produced were maximum (66 g/L) on the fifth day of fermentation that was carried out

at 37°C, initial pH adjustment at 7 and enzymatic pretreatment with the yield of 0.723 g

organic acids/g initial COD. The main organic acids produced was acetic acid (50%),

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followed by lactic acid (33%), iso-butyric acid (9%) and n-butyric acids (8%). The

results indicate that high concentration of organic acids were produced in anaerobic

treatment of restaurant wastes, which could served as alternative and abundant substrate

for the production of biodegradable plastics. The supernatant collected from the

anaerobic treatment of highest organic acids production was concentrated by vacuum

evaporation to be used for PHB production. The total organic acids obtained after

evaporation process was about 100 g/L. In the second stage, the concentrated organic

acids were successfully converted to PHB using batch and pH-stat fed-batch

fermentation by recombinant Escherichia coli containing. In batch fermentation about

6.8 g/L of cell dry weight and 2.5 g/L of PHB were achieved after 18 hours of

cultivation, resulting in PHB content of 36% and productivity of PHB 0.14 g L-1 h-1. On

the other hand, the maximum PHB concentration obtained was 9.2 g/L after 17 hours

corresponding to 44% (g/g) of PHB content and 0.539 g L-1 h-1 productivity in pH-stat

fed-batch fermentation.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

PENGHASILAN ASID ORGANIK DARIPADA SISA BUANGAN RESTORAN SEBAGAI SUBSTRAT UNTUK PENGHASILAN POLYHYDROXYBUTYRATE

OLEH REKOMBINAN E. COLI

Oleh

MAJD ESHTAYA

Jun 2007

Pengerusi: Profesor Mohd Ali Hassan, PhD

Fakulti: Kejuruteraan

Di dalam kajian ini, bahan buangan restoran telah digunakan sebagai sumber karbon

tunggal untuk penghasilan polyhydroxybutyrate (PHB) oleh rekombinan Escherichia

coli. Penghasilan PHB dilakukan melalui dua peringkat; penghasilan asid organik

daripada bahan buangan restoran yang dirawat seterusnya penghasilan PHB

menggunakan asid organik tersebut. Dalam peringkat pertama, kesan pH permulaan

fermentasi, suhu operasi and pra-rawatan berenzim (hidrolisis menggunakan α-amylase

dan glucoamylase) telah dikaji untuk menggalakkan penghasilan asid organik daripada

bahan buangan restoran dalam rawatan anaerobik. Rawatan anaerobik telah dijalankan

dalam kelalang Erlenmeyer menggunakan inkubator penggoncang orbital. Tiga suhu

yang berbeza telah dipilih; 30°C, 37°C dan tanpa kawalan (suhu bilik). pH permulaan

proses fermentasi juga berbeza; pH 7 dan tanpa pembetulan pH. Maksimum asid organik

yang dihasilkan ialah 66 g/L pada hari kelima fermentasi yang dijalankan pada suhu

37°C, pH permulaan 7, dan pra-rawatan berenzim memberikan angkali hasilan 0.723 g

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asid organik/COD awal. Asid organik utama yang dihasilkan ialah asetik (50%), diikuti

oleh asid laktik (33%), asid iso-butirik (9%) dan asid n-butirik (8%). Keputusan tersebut

menunjukkan asid organik pada kepekatan yang tinggi dapat dihasilkan dalam pra-

rawatan bahan buangan restoran, dimana ia boleh membekalkan substrat yang murah

dan banyak untuk penghasilan plastik boleh urai. Supernatan yang dikumpul daripada

pra-rawatan yang paling tinggi penghasilan asid organik telah dipekatkan menggunakan

pengewap kedap udara untuk digunakan seterusnya dalam peringkat penghasilan PHB.

Jumlah asid organik yang dihasilkan selepas proses pengewapan adalah sekitar 100 g/L.

Dalam peringkat kedua, asid organik yang dipekatkan telah berjaya ditukarkan kepada

PHB menggunakan fermentasi sekelompok dan suapan sesekelompok oleh rekombinan

E.coli. Dalam fermentasi sekelompok, 6.8 g/L berat sel kering dan 2.5 g/L PHB telah

dihasilkan selepas inokulasi selama 18 jam menghasilkan kandungan PHB dalam sel

sebanyak 36% dan produktiviti PHB ialah 0.14 g L-1 h-1. Sebaliknya bagi fermentasi

suapan sesekelompok pH-stat, kepekatan maksimum PHB yang didapati adalah 9.2 g/L

selepas 72 jam fermentasi bersamaan dengan 44% g/g kandungan PHB dan 0.539 g L-1

h-1 produktiviti.

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ACKNOWLEDGEMENTS

Allhamdulillah, thanks a lot to merciful Allah for giving me the strength to finish this

project.

I would like to express my appreciation and gratitude to my chairman Professor Mohd.

Ali Hassan and co-supervisor Dr. Aini binti Abdul Rahman for guidance, suggestion and

encouragement throughout this project.

My gratitude and thanks to my laboratory mates, Chong Mei Ling, Hidayah Ariffin,

Tabassum Mumtaz, Sharifah Soplah Syed Abdullah, Mohd Huzairi Mohd Zainuddin,

Asma’ Hashim, Alawi Hj. Sulaiman, Mohd Zulkhairi Mohd Yusoff for their valuable

discussion, help and cooperation in this project.

I gratefully acknowledge the financial support by Islamic Development Bank (IDB).

Also my heartiest thanks to all my friends in Palestine and Malaysia for their friendship,

support and encouragement throughout my study.

I am sincerely grateful to my parents, my sisters and my brothers for their love, endless

support, encouragement, understanding and reassurance during my graduate degree.

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I certify that an Examination Committee met on 15 June 2007 to conduct the final examination of Majd Khalid Eshtaya on her Master of Science thesis entitled “Production of Organic Acids from Restaurant Wastes as Substrate for Polyhydroxybutyrate (PHB) by Recombinant Escherichia Coli” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as Follows:

CHIN NYUK LING, PhD Faculty of Engineering Universiti Putra Malaysia (Chairman)

SITI MAZLINA MUSTAFA KAMAL, PhD Faculty of Engineering Universiti Putra Malaysia (Internal Examiner)

SURAINI ABD. AZIZ, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner)

AWANG BONO, PhD Associate Professor Faculty of Graduate Studies Universiti Malaysia Sabah (External Examiner)

HASANAH MOHD. GHAZALI, PhD Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date:

viii

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Master of Science. The members of Supervisory Committee are as follows:

Mohd. Ali Hassan, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)

Nor’Aini Abdul Rahman, PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)

AINI IDERIS, PhD Professor/Dean School of Graduate Studies Universiti Putra Malaysia

Date: 9 August 2007

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DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citation which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

MAJD ESHTAYA

Date: July 2007

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TABLE OF CONTENTS

Page

DEDICATION ii ABSTRACT iii ABSTRAK v ACKNOWLEDGEMENTS vii APPROVAL viii DECLARATION x LIST OF TABLES xiv LIST OF FIGURES xv LIST OF ABBREVIATIONS/NOTATIONS/GLOSSARY OF TERMS xvi CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 4 2.1 Conventional Plastics 4 2.2 Polyhydroxyalkanoates (PHAs) 6

2.2.1 General Structure of PHA and Some Representative Members 6

2.2.2 Pathways of PHB Synthesis 8 2.2.3 PHA Production from Inexpensive Carbon

Substrates 10 2.2.4 PHA Production by Recombinant Escherichia coli 11 2.2.5 Properties of PHB 15 2.2.6 Biodegradability of PHAs 17 2.2.7 Applications and Commercialization 18 2.2.8 Recovery of PHA 22

2.3 Municipal Solid Waste (MSW) 23 2.3.1 Generation and Composition 24 2.3.2 The Solid Waste Management Hierarchy 26

2.4 Food Waste 26 2.5 The Anaerobic Digestion Process 27

2.5.1 Stages in Anaerobic Digestion 28 2.5.2 Important Operating Parameters in Anaerobic

Digestion Process 32

xi

3 PRODUCTION OF ORGANIC ACIDS FROM RESTAURANT WASTES BY ANAEROBIC DIGESTION 36 3.1 Introduction 36 3.2 Materials and Methods 38

3.2.1 Characteristic of Restaurant Wastes 38 3.2.2 Batch Anaerobic Fermentation 39

3.3 Analytical Methods 39 3.3.1 Organic Acids Determination 39 3.3.2 Total Sugars (Phenol-sulfuric acid method) 40 3.3.3 Proximate analysis 40 3.3.4 Total Solids 40 3.3.5 Total Nitrogen 41 3.3.6 Chemical Oxygen Demand (COD) 41 3.3.7 Assay of Glucoamylase Activity 41 3.3.8 Assay α-Amylase Activity 42

3.4 Results and Discussion 42 3.4.1 Characteristic of Food Wastes 42 3.4.2 Effect of Temperature 44 3.4.3 Effect of Initial pH 49 3.4.4 Effect of Pretreatment Process 53

3.5 Conclusion 58

4 PHB PRODUCTION USING ORGANIC ACIDS PRODUCED FROM RESTAURANT WASTES BY RECOMBINANT E.coli 59 4.1 Introduction 59 4.2 Materials and Methods 60

4.2.1 Chemical Reagents 60 4.2.2 Microorganism and Culture Conditions 61 4.2.3 Flask Culture 62 4.2.4 Batch and Fed-Batch Production of PHB 62

4.3 Analytical Methods 64 4.3.1 PHB Determination by HPLC 64 4.3.2 Cell Dry Weight (CDW) 65 4.3.3 Organic Acids from Restaurant Wastes 65

4.4 Results 66 4.4.1 PHB Production in Flask Culture 66 4.4.2 PHB Production in Batch Culture Using

Different Substrate 67 4.4.3 PHB Production Using Fed-Batch Culture 72

4.5 Discussion 74 4.6 Conclusion 78

5 DISCUSSION, CONCLUSION AND SUGGESTION FOR

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FUTURE STUDIES 79 5.1 General Discussion 79 5.2 Conclusion 81 5.3 Suggestions for Future Work 82

REFERENCES 84 APPENDICES 90 BIODATA OF THE AUTHOR 102

xiii

LIST OF TABLES

Table Page 2.1 Comparison of several PHAs production process in recombinant E. coli 14 2.2 Physical properties of various PHAs and polypropylene 16 2.3 Possible applications of PHAs 19 2.4 Manufacturers, microorganisms, and the raw materials used for the production of

biodegradable plastics 21 2.5 Typical composition of MSW in developed countries 25 2.6 Composition of MSW in selected Asian countries (%) 25 3.1 Characteristics of food waste 43 3.2 Summary of organic acids produced in different treatments of restaurant wastes

from anaerobic treatment 57 4.1 Comparison of PHB production from glucose, synthetic organic acids and organic

acids obtained from restaurant wastes fermentation in flask culture 67 4.2 Comparison of PHB production from glucose, synthetic organic acids and organic

acids obtained from restaurant wastes fermentation in batch culture 72

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LIST OF FIGURES

Figure Page 2.1 General structure of polyhydroxyalkanoates and some representative members 7 2.2 Metabolic pathway involved in the synthesis of PHB. Hatched arrows indicate

negative regulatory effects 9 2.3 Schematic of the different metabolic steps and microbe groups involved in the

complete degradation of organic matter to methane and carbon dioxide 29 3.1 Organic acids produced during anaerobic treatment of restaurant waste without

initial pH adjustment and pretreatment with addition of water at ratio (1:1). (a) 30°C (b) 37°C (c) uncontrolled temperature (room temperature 21-25°C) 46

3.2 Organic acids produced during anaerobic treatment of food waste at 37°C and

pretreatment with addition of water at ratio (1:1) (a) without initial pH adjustment (b) adjustment initial pH at 7 50

3.3 Effect of pretreatment process on organic acids produced during anaerobic

treatment of food waste with initial pH adjustment to 7 and temperature 37 °C 55

4.1 Schematic description of bioreactor for PHB production. 63 4.2 Time course of PHB production by recombinant E.coli using glucose as carbon

substrate in batch culture 68 4.3 Time course of PHB production by recombinant E.coli using synthetic organic

acids as carbon substrate in batch culture 69 4.4 Time course of PHB production by recombinant E.coli using organic acids

obtained from restaurant wastes fermentation as carbon substrate in batch culture 71

4.5 Time course of PHB production by recombinant E.coli using organic acids

obtained from restaurant wastes fermentation as carbon substrate in fed-batch culture 73

xv

LIST OF ABBREVIATIONS

CDM Clean Development Mechanism

CDW Cell dry weight

COD Chemical oxygen demand

Da Dalton

GC Gas chromatography

HPLC High performance liquid chromatography

HRT Hydraulic retention time

MCL Medium Chain Length

MSW Municipal solid waste

NADH Nicotinamide Adenine Dinucleotide

NADPH Nicotinamide Adenine Dinucleotide Phosphate

(reduced form)

OD Optical density

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

P(4HB) Poly (4-hydroxybutyrate)

P(HBHHx) Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate

PHA Polyhydroxyalkanoate

PHB Polyhydroxybutyrate

PHO Poly (3-hydroxyoctanoate)

PHV Polyhydroxyvalerate

rpm Rotation per minute

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SCL Short Chain Length

TKN Total Kjeldahl Nitrogen

VFA Volatile Fatty Acid

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xviii

CHAPTER 1

INTRODUCTION

Over the past decades, plastics derived from petrochemicals, have become indispensable

materials. Unfortunately, most of these materials are not biodegradable, thus they remain

indefinitely in landfill sites and this causes serious environmental and waste

management problems. On the other hand, when plastic waste are disposed of by

incineration, numerous air pollution problems are generated from the incineration of the

chemical additives in plastic formulations such as ash disposal, emission of dioxins, acid

gases, carbon dioxide and other toxic gases (Wang et al., 2003).

Study by Khanna and Srivastava (2005) discussed about the problems concerning the

global environment and solid waste management have created much interest in the

development of biodegradable polymers as replacement to non-biodegradable

petrochemicals plastics. Among the several biodegradable polymers under

development, polyhydroxyalkanoates (PHAs) have attracted much attention because of

their similar material properties to the conventional petrochemical derived plastics and

their complete biodegradability in various environments. Polyhydroxyalkanoates

(PHAs) are polyesters of hydroxyalkanoates that are synthesized and intracellularly

accumulated as an energy and/or carbon storage material by numerous microorganisms.

Polyhydroxybutyrate (PHB) was the first PHA to be discovered and is the most widely

studied and best characterized PHA. It is accumulated as a membrane enclosed inclusion

in many bacteria at up to 80% of the dry cell weight. It has mechanical properties very

similar to conventional plastics like polypropylene.

One of the major drawbacks of employing PHAs in a wide range of applications is the

high production cost, which makes it difficult to compete with petrochemical-based

polymers. Therefore, much effort has been devoted to reducing the production cost of

PHAs by the development of better bacterial strains and fermentation that is more

efficient and recovery processes. Recombinant Escherichia coli strain, which has been

thought to be a potential host for the production of PHAs, has been successfully

employed for the production of PHB to a high concentration with high productivity. One

of the important factors in determining the applicability of PHAs production on the

industrial scale is the cost of raw materials (Ahn et al., 2001). In recent years, there has

been a growing interest in the production of biodegradable plastics from renewable

resources such as municipal solid waste, wastes from the food industry, household and

waste from agriculture and forestry as a cheaper carbon substrate (Wang et al., 2003).

In this study, restaurant waste is used as the raw material for PHB production since food

waste is a nutrition-rich resource and it can be used as a substrate in fermentation.

Therefore, it is possible to consider coupling a restaurant waste treatment process with

PHB production. This technique is beneficial in several ways. Firstly, it can reduce the

quantity of restaurant waste, therefore reducing the costs of food waste treatment;

secondly, large amount of organic acids (lactic, acetic, proionic and butyric acids)

produced during anaerobic treatment of restaurant waste is used to produce PHB by

recombinant Escherichia coli containing the Ralstonia eutropha PHB biosynthetic

genes.

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The objectives of this study are as follows:

1. The production of organic acids from restaurant waste in anaerobic treatment at

various conditions.

2. To utilize the organic acids obtained from fermented restaurant waste as

substrates for the production of polyhydroxybutyrate (PHB) by recombinant

Escherichia coli containing in batch and fed-batch culture.

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CHAPTER 2

LITERATURE REVIEW

2.1 Conventional Plastics

Within the last 50 years, petrochemical plastics have become one of our most applied

materials. Plastics have wide range of strengths and shapes, lightness, low price,

durability and resistance to degradation (Khanna and Srivastava, 2005) and the

application of plastics in industry ranging from automobiles to medical (Zinn et al.,

2001).

The problem of environmental pollution caused by dumping of plastic waste has

assumed global proportions. Plastic waste, which accounts approximately 20–30% of the

volume of municipal solid waste landfill sites are recalcitrant to microbial degradation

(Ishigaki et al., 2004). Due to very fast development of society and the limitation of

satisfactory landfill sites burial of plastics wastes in landfill is a time bomb (Suprakas

and Bousmina, 2005). Solutions to plastic waste management include source reduction,

incineration, recycling and bio- or photo-degradation (Khanna and Srivastava, 2005).

Incineration plastics wastes are dangerous and expensive. During the combustion of

plastic waste, a large amount of carbon dioxide produces and creates global warming

and some times produces toxic gases like hydrogen chloride and hydrogen cyanide,

which again contribute to global pollution. Furthermore, recycling has some major

4

disadvantages, as it is difficult sorting the wide variety of plastics and there are changes

in the plastic’s material such that its further application range is limited (Reddy et al.,

2003).

On these backgrounds, there is an urgent need for the development of friendly polymeric

materials that would not involve the use of toxic or noxious component in their

manufacture and could be degradable in the natural environmental conditions. For these

reasons, throughout the world today, the development of biodegradable materials with

controlled properties has been a subject of great research challenge to the community of

materials scientists and engineers (Suprakas and Bousmina, 2005).

Three types of biodegradable plastics introduced are photodegradable, semi

biodegradable and completely biodegradable. Photodegradable plastics have light

sensitive groups incorporated directly into the backbone of the polymer as additives.

However, landfills lack sunlight and thus they remain non-degraded. Semi-

biodegradable plastics are the starch-linked plastics where starch is incorporated to hold

together short fragments of polyethylene. Soil microorganisms degrade the starch easily,

thus breaking down the polymer matrix while the polyethylene fragments remain non-

degradable. The third type of biodegradable plastics is utilization by bacteria to form a

biopolymer these include polyhydroxyalkanoates (PHA), polylactides (PLA), aliphatic

polyesters, polysaccharides, copolymers and/or blends of the above (Reddy et al., 2003).

Among the various biodegradable polymer materials, polyhydroxyalkanoates (PHAs)

are attractive substitutes for conventional petrochemical plastics because of their similar

material properties to conventional plastics and complete biodegradability after disposal

5

(Ojumu et al., 2004).

2.2 Polyhydroxyalkanoates (PHAs)

Polyhydroxyalkanoates (PHAs) are biological polyesters of various hydroxyalkanoates,

which are accumulated as a carbon and/or energy storage material in various

microorganisms usually under the condition of limiting nutritional elements such as

nitrogen, sulphur, phosphate, oxygen, magnesium and potassium in the presence of

excess carbon source. After the first discovery of poly (3-hydroxybutyrate) (PHB) from

the bacterium Bacillus megaterium by the French scientist Lemoigne in 1926, many

different PHAs possessing different numbers of main chain carbon atoms and different

types of pendent groups have been found (Lee and Park, 2005). However only several

PHA including PHB, copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate P(3HB-

co-3HV), poly 4-hydroxybutyrate P(4HB), copolymers of 3-hydroxybutyrate and 3-

hydroxyhexanoate (PHBHHx) and poly 3-hydroxyoctanoate (PHO) are available in

sufficient quantity for application or research purpose (Chen and Wu, 2005).

2.2.1 General Structure of PHA and Some Representative Members

More than 100 different monomer units have been identified as constituents of the

storage PHA as shown in Figure 2.1. This enables producing different types of

biodegradable polymers with an extensive range of properties. The monomer units are

all in D (-) configuration due to the stereo-specificity of the biosynthetic enzymes which

is important for their biodegradability and biocompatibility (Reddy et al., 2003).

6