Biodegradable Poly(3-hydroxybutyrate-co-3-hydroxyvalerate

Biodegradable Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and Renewable

Biomass based Composites and Marine Degradation Behaviour

by

Kjeld Meereboer

A Thesis

presented to

The University of Guelph

In partial fulfilment of requirements

for the degree of

Master of Applied Science

in

Engineering

Guelph, Ontario, Canada

© Kjeld Meereboer, May, 2020

ABSTRACT

BIODEGRADABLE POLY(3-HYDROXYBUTYRATE-CO-3-HYDROXYVALERATE) AND

RENEWABLE BIOMASS BASED COMPOSITES AND MARINE DEGRADATION

BEHAVIOUR

Kjeld Meereboer

University of Guelph, 2020

Advisor(s):

Dr. Manjusri Misra

Dr. Amar K. Mohanty

This thesis involves an investigation on developing biodegradable poly(3-hydroxybutyrate-co-3-

hydroxyvalerate) (PHBV) based blends, and biocomposites. PHBV is derived from various

agricultural carbon sources and was extruded with plasticized cellulose acetate (pCA), to develop

a sustainable and biodegradable PHBV/pCA blend. PHBV/pCA blends were found to be

immiscible and had reduced mechanical performance and flexibility compared to the individua l

components. Miscanthus fibre and distillers dried grains with solubles (DDGS) biocomposites of

PHBV were developed to ascertain the effect on marine biodegradability. Under ASTM D7991-

15, PHBV/Misc (75/25) and PHBV/DDGS (75/25) biocomposites increased the biodegradation

rate of PHBV by 24 and 40% respectively. Marine biodegradation of PHBV/DDGS (75/25)

composite exceeded cellulose and is 100% marine biodegradable in 260 days, attributed to the

presence of cellulosic material and protein promoting microbial growth in DDGS. PHBV/Misc

(85/15) and (75/25) had increased tensile/flexural moduli by 55 and 100%, and impact strength of

100%, relative to PHBV.

iii

ACKNOWLEDGEMENTS

I would like to thank Dr. Manjusri Misra and Amar K. Mohanty for taking me on as a

Master’s student and believing I had potential to succeed in the area of polymer sciences. They

have provided me with an unforgettable opportunity to learn amongst this prestigious research

group. Along with their guidance I know I have left my mark in the area of biodegradable polymer

research from today onwards and I will walk away with a higher understanding of my own impact

on the environment.

I would especially like to thank those at the Bioproducts Discovery and Development

Centre (BDDC) for giving me this opportunity with prudent support in all aspects of research and

social growth. Without the members at the BDDC; fellow associates, postdocs, PhD’s, Masters

and undergraduates, this experience would not have been as wonderful as it became. When walking

away from here I know I will always have advisors and friends that I shared this experience with.

I would also like to thank to my advisory committee member Dr. Loong-Tak Lim for

pushing me towards, and supporting me, in the area food packaging and engineering. I would also

like to acknowledge Prof. Ping Wu’s support throughout my degree is something I will look back

on with much appreciation.

Finally, I would like to thank my family for giving some much needed reprieve from work

and love I sometimes took for granted; those with me now and the one that left me recently, during

my graduate studies.

I would like to thank the following financial supporters of this research: Natural Sciences

and Engineering Research Council (NSERC), Canada Discovery Grants (Project # 400320); the

Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) – University of Guelph, the

Bioeconomy Industrial Uses Research Program Theme (Project # 030054, 030177, 030255 and

030361); OMAFRA-University of Guelph Gryphon’s Leading to the Accelerated Adoption of

Innovative Research (LAAIR) Program (Project # 298635); and the Ontario Research Fund,

Research Excellence Program; Round-9 (ORF-RE09) from the Ontario Ministry of Economic

Development, Job Creation and Trade, Canada (Project #053970 and # 054345).

iv

My research has also benefited from the facility funding to the BDDC supported by

FedDev Ontario; Ontario Ministry of Agriculture, Food, and Rural affairs (OMAFRA); Canada

Foundation for Innovation (CFI); Federal Post-Secondary Institutions Strategic Investment Fund

(SIF); and matching funds from the province of Ontario, Bank of Montreal (BMO) and numerous

University of Guelph's Alumni.

v

TABLE OF CONTENTS

Abstract ............................................................................................................................................ ii

Acknowledgements ......................................................................................................................... iii

Table of Contents .............................................................................................................................v

List of Tables ...................................................................................................................................x

List of Figures ................................................................................................................................. xi

List of Abbreviations .................................................................................................................... xiii

List of Publications ........................................................................................................................ xv

Chapter 1: Introduction .................................................................................................................. 1

1.1 Structure of Thesis and Connecting Statements............................................................... 1

1.2 Problem Statement ........................................................................................................... 2

1.3 Objectives and Hypothesis ............................................................................................... 3

1.3.1 Objective 1: PHBV Blending.................................................................................... 3

1.3.2 Hypothesis 1.............................................................................................................. 3

1.3.3 Objective 2: Biodegradation of PHBV/Miscanthus Biocomposites ......................... 3

1.3.4 Hypothesis 2.............................................................................................................. 3

1.3.5 Objective 3: Biodegradation of PHBV/DDGS Biocomposites ................................ 4

1.3.6 Hypothesis 3.............................................................................................................. 4

1.4 Significance ...................................................................................................................... 4

Chapter 2: Literature Review ......................................................................................................... 6

2.1 Introduction ...................................................................................................................... 8

2.1.1 Polymer Pollution in the Environment ..................................................................... 8

2.2 Bacterial-Polyesters........................................................................................................ 13

2.2.1 PHAs ....................................................................................................................... 15

2.2.2 Challenges of Biodegradable Plastics ..................................................................... 18

2.3 What is Biodegradation? ................................................................................................ 20

2.3.1 Aerobic vs Anaerobic Biodegradation .................................................................... 25

2.3.2 Misinterpretation of Oxo-degradable Plastics ........................................................ 26

vi

2.3.3 Aerobic Biodegradation Standards for Polymers and Their Limits ........................ 29

2.3.4 Anaerobic Biodegradation Standards for Polymers and Their Limits .................... 33

2.4 PHA Biodegradation ...................................................................................................... 35

2.4.1 PHA Biodegrading Microorganisms....................................................................... 36

2.4.2 Extracellular PHA Biodegradation ......................................................................... 36

2.4.3 PHA Attributes that Affect Biodegradation............................................................ 38

2.4.4 Chemical Additives and Blending Effect on PHA Biodegradation ........................ 44

2.4.5 PHA Soil Biodegradation ....................................................................................... 47

2.4.6 PHA Composting .................................................................................................... 53

2.4.7 PHA Marine Biodegradation .................................................................................. 56

2.4.8 PHA Sewage Sludge Biodegradation ..................................................................... 60

2.4.9 PHA Anaerobic Digestion Biodegradation............................................................. 62

2.4.10 PHA Accelerated Landfill Biodegradation ............................................................. 64

2.4.11 Conclusions of Biodegradation ............................................................................... 65

2.5 PHA-Based Biocomposites ............................................................................................ 66

2.5.1 Natural Fibres and fillers......................................................................................... 66

2.5.2 Compatibilizers/Coupling Agents........................................................................... 67

2.6 PHA-Based Composites Biodegradation ....................................................................... 69

2.6.1 Composite Soil Biodegradation .............................................................................. 71

2.6.2 Composite Composting........................................................................................... 76

2.6.3 Composite Marine Biodegradation ......................................................................... 78

2.6.4 Conclusions of Composite Biodegradation ............................................................ 80

2.7 Conclusion...................................................................................................................... 80

2.8 References ...................................................................................................................... 82

Chapter 3: Sustainable PHBV/Cellulose Acetate Blends: Effect of Chain Extender and

Plasticizer .................................................................................................................................... 121

3.1 Introduction .................................................................................................................. 122

3.2 Materials and Methods ................................................................................................. 125

3.2.1 Materials................................................................................................................ 125

3.2.2 Preparation of Plasticized Cellulose Acetate ........................................................ 126

vii

3.2.3 Melt Extrusion Followed by Injection Moulding ................................................. 126

3.2.4 Solubility Calculations .......................................................................................... 127

3.2.5 Differential Scanning Calorimetry (DSC) ............................................................ 127

3.2.6 Thermogravimetric Analysis (TGA)..................................................................... 128

3.2.7 Heat Deflection Temperature (HDT) .................................................................... 128

3.2.8 Dynamic Mechanical Analysis (DMA) ................................................................ 128

3.2.9 Tensile and Flexural Properties............................................................................. 129

3.2.10 Notched IZOD Impact Strength ............................................................................ 129

3.2.11 Density .................................................................................................................. 129

3.2.12 Scanning Electron Microscopy (SEM) ................................................................. 129

3.3 Results and Discussion................................................................................................. 130

3.3.1 Solubility Parameters ............................................................................................ 130

3.3.2 Thermal Properties ................................................................................................ 130

3.3.3 DMA ..................................................................................................................... 136

3.3.4 Mechanical Properties........................................................................................... 138

3.3.5 HDT and Density .................................................................................................. 141

3.3.6 SEM ...................................................................................................................... 142

3.4 Conclusions .................................................................................................................. 145

3.5 References .................................................................................................................... 145

Chapter 4: Marine Degradation Behaviour of PHAs and Natural Fibres-based Biocomposites151

4.1 Introduction .................................................................................................................. 152


4.2.1 Materials................................................................................................................ 156

4.2.2 Composite Processing ........................................................................................... 157

4.2.3 Elemental Analysis (CHNS) ................................................................................. 157




4.2.7 Contact Angle ....................................................................................................... 159

viii

4.2.8 Marine Biodegradation ......................................................................................... 159



4.3.2 DSC ....................................................................................................................... 161

4.3.3 TGA ...................................................................................................................... 163

4.3.4 Elemental Analysis ............................................................................................... 163

4.3.5 SEM Morphology ................................................................................................. 164

4.3.6 Contact Angle ....................................................................................................... 165


4.4 Conclusion.................................................................................................................... 167

4.5 References .................................................................................................................... 168

Chapter 5: Marine Degradation Behaviour of PHAs and Distillers Dried Grains with Solubles

Biocomposites ............................................................................................................................. 173

5.1 Introduction .................................................................................................................. 174


5.2.1 Materials................................................................................................................ 177

5.2.2 Composite Processing ........................................................................................... 177

5.2.3 Elemental Analysis (CHNS) ................................................................................. 177




5.2.7 Contact Angle ....................................................................................................... 179




5.3.2 DSC ....................................................................................................................... 181

5.3.3 TGA ...................................................................................................................... 182

5.3.4 Elemental Analysis ............................................................................................... 183

5.3.5 SEM Morphology (To be completed and SEM of samples)................................. 184

5.3.6 Contact Angle ....................................................................................................... 185

ix


5.4 Conclusion.................................................................................................................... 187

5.5 References .................................................................................................................... 188

Chapter 6: Overall Conclusions and Future Work..................................................................... 194

6.1 Overall Conclusions ..................................................................................................... 194

6.2 Future Work ................................................................................................................. 196

x

LIST OF TABLES

Table 2.1: PHA Soil Biodegradation Studies from 2010-2020. ................................................... 48

Table 2.2: PHA Composting Biodegradation Studies from 2010-2020. ...................................... 53

Table 2.3: Recent PHA Marine Biodegradation Studies. ............................................................. 56

Table 2.4: PHA Anaerobic Sewage Sludge Biodegradation Studies. ........................................... 60

Table 2.5: PHA Composite Soil Biodegradation Studies from 2010-2020. ................................. 72

Table 2.6: PHA Composite Composting Studies from 2010-2020............................................... 78

Table 2.7: PHA Composite Marine Biodegradation Studies. ....................................................... 79

Table 3.1. PHBV/CA blend compositions. ................................................................................. 126

Table 3.2. Solubility Parameters (δ) of CA, PHBV and TEC. ................................................... 130

Table 3.3. DSC results of PHBV/PCA blends with and without 0.3 phr CE. ............................ 133

Table 3.4. Glass transition temperatures of PHBV/pCA blends with and without CE measured by

DMA analysis. ............................................................................................................................ 138

Table 3.5. Heat deflection temperature and density of PHBV/pCA/CE blends. ........................ 142

Table 4.1: Marine water initial parameters ................................................................................. 159

Table 4.2: DSC of PHBV/Misc composites. .............................................................................. 162

Table 4.3: TGA of marine biodegradation samples, Miscanthus and cellulose. ........................ 163

Table 4.4: Elemental analysis of marine biodegradation samples and sediment. ....................... 164

Table 4.5: Contact angles of polymer composites and cellulose filter paper. ............................ 165

Table 4.6: Marine biodegradation results. .................................................................................. 167

Table 5.1: Marine Water Parameters. ......................................................................................... 180

Table 5.2: DSC of PHBV/DDGS Composites. ........................................................................... 182

Table 5.3: TGA of Marine Biodegradation Samples, DDGS and Cellulose. ............................. 183

Table 5.4: Elemental Analysis of Marine Biodegradation Samples and Sediment. ................... 184

Table 5.5: Contact Angles of Polymer Composites and Cellulose Filter Paper. ........................ 185

Table 5.6: Marine Biodegradation Results. ................................................................................ 187

xi

LIST OF FIGURES

Figure 2.1: Production Energy and CO2 Emissions of Petroleum-based and Bio-based Polymers 6,8. .................................................................................................................................................... 9

Figure 2.2: Predominant Current Polymer Waste Disposal Streams 9.......................................... 10

Figure 2.3: Cradle-to-Cradle and Cradle-to-Grave Approach of Biodegradable and Non-

Biodegradable Plastic.................................................................................................................... 15

Figure 2.4: a) 3-hydroxypropionate (HP), b) 3-hydroxybutyrate (HB), c) 3-hydroxyvalerate

(HV), d) 4-hydroxybutyrate (4HB), e) 3-hydroxyhexanoate (Hx), f) 3-hydroxyoctonaote (HO)

chemical structures........................................................................................................................ 16

Figure 2.5: Young's Modulus and Elongation at Break of Plastic Types. Reprinted (adapted) with

permission (Creative Commons) from Rastogi et al. (2015) on April 29, 2020 86. ...................... 20

Figure 2.6: Basic Degradation Pathways of Polymers. ................................................................. 22

Figure 2.7: Bulk Erosion and Surface Erosion Effects on Polymers as Function of Time.

Redrawn with Permission (Lic. #4818250646220) from Van Dijkhuizen-Radersma et al. (2008)

on April 29, 2020 97. ..................................................................................................................... 23

Figure 2.8: Theoretical Biodegradation Pathway of Polymer Material. ....................................... 24

Figure 2.9: ASTM D6954-18 Oxidation and Biodegradation Standard Test Procedure 120. ........ 28

Figure 2.10: Marine Biodegradation and Degradation ASTM standards. .................................... 33

Figure 2.11: Single PHB Crystal Enzymatic Degradation by PHB Depolymerase. Reprinted with

permission (Lic. #4786571333526) from Iwata et al. (1999) on March 12, 2020 162. ................. 38

Figure 2.12: a) PHB and PHBV crystallinity from WAXD patterns. b) rate of weight loss by 2µg

of Ralstonia pickettii type 1 PHA depolymerase due to 3HV ratio. Reprinted (adapted) with

permission (Lic. #4786580163872) from Feng et al. (2004) on March 12, 2020 167. .................. 39

Figure 2.13: Schematic model of enzymatic degradation behaviour of lamellar crystal in P(HB)

solution-grown single crystal by PHB depolymerase. Reprinted (adapted) with permission from

(Numata et al. H. Enzymatic Degradation Processes of Poly[(R)-3-Hydroxybutyric Acid] and

Poly[(R)-3-Hydroxybutyric Acid-Co-(R)-3-Hydroxyvaleric Acid] Single Crystals Revealed by

Atomic Force Microscopy: Effects of Molecular Weight and Second-Monomer Composition on

Erosion) on March 12, 2020. Copyright (2005) American Chemical Society 165. ....................... 41

Figure 2.14: PHBV Film Surface at 35 days in Composting Conditions. Reprinted with

permission (Lic. #4786580681302) from Weng et al. (2010) on March 12, 2020 176. ................. 44

Figure 2.15: Basic Natural Fibre Structure. .................................................................................. 67

Figure 2.16: Reaction scheme between PHBV and MA in presence of an initiator. Redrawn with

permission (Lic. #4786581071720) from Avella et al. (2007) on March 12, 2020 261................. 68

Figure 3.1. DSC second heating curves of (A) PHBV/pCA blend ratios, and (B) PHBV/pCA/CE

blends with 0.3 phr CE and DSC of cooling curve of (C) PHBV/pCA blend ratios, and (D)

PHBV/pCA/CE blends with 0.3 phr CE. .................................................................................... 132

xii

Figure 3.2. TGA (A) and DTGA (B) of PHBV/pCA blends and TGA (C) and DTGA (D) of

PHBV/pCA/CE blends with 0.3 phr CE. .................................................................................... 135

Figure 3.3: TGA (A) and DTGA (B) of TEC, pCA and CA. ..................................................... 136

Figure 3.4. Storage modulus of (A) PHBV/pCA blends and (B) PHBV/pCA/CE blends with 0.3

phr CE, and Tan(δ) of (C) PHBV/pCA blend ratios and (D) PHBV/pCA/CE blends with 0.3 phr

CE................................................................................................................................................ 137

Figure 3.5. Tensile modulus and strength of PHBV/pCA blends: (A) 100/0, (B) 70/30, (C) 50/50,

(D) 30/70, (E) 0/100, (F) 70/30 + 0.3phr CE, (G) 50/50 + 0.3phr CE, and (H) 30/70 + 0.3phr CE.

..................................................................................................................................................... 140

Figure 3.6. Flexural modulus and strength of PHBV/pCA blends: (A) 100/0, (B) 70/30, (C)

50/50, (D) 30/70, (E) 0/100, (F) 70/30 + 0.3phr CE, (G) 50/50 + 0.3phr CE, and (H) 30/70 +

0.3phr CE. ................................................................................................................................... 140

Figure 3.7. Elongation at break and notched IZOD impact strength of PHBV/pCA blends: (A)

100/0, (B) 70/30, (C) 50/50, (D) 30/70, (E) 0/100, (F) 70/30 + 0.3phr CE, (G) 50/50 + 0.3phr CE,

and (H) 30/70 + 0.3phr CE. ........................................................................................................ 141

Figure 3.8. SEM morphology of PHBV/pCA/CE blend ratios: (A) 100/0, (B) 70/30, (C) 50/50,

(D) 30/70, (E) 0/100, (F) 70/30 + 0.3phr CE, (G) 50/50 + 0.3phr CE, and (H) 30/70 + 0.3phr CE

after break. .................................................................................................................................. 144

Figure 4.1: Articles published in the area of polymer science, on the topic of "marine degradation

of polymer" from Web of Science (excluding review articles) (Obtained February 11, 2020). . 153

Figure 4.2: Expected pathway of PHBV degradation 20. ............................................................ 155

Figure 4.3: PHBV/Misc A) tensile modulus and strength, B) flexural modulus and strength, and

C) elongation at break and impact strength. ............................................................................... 161

Figure 4.4: Notched IZOD Cross-Section SEM Morphology of A) PHBV, B) PHBV/Misc 85/15

and C) PHBV/Misc 75/25 impact samples after break. .............................................................. 165

Figure 4.5: Cellulose and PHBV/Misc biocomposites A) CO2 evolution and B) overall

biodegradation............................................................................................................................. 167

Figure 5.1: Proteinaceous Filler Bio-assimilation by Microorganisms 27................................... 176

Figure 5.2: PHBV/DDGS A) Tensile Modulus and Strength, B) Flexural Modulus and Strength,

and C) Elongation at Break and Impact Strength. ...................................................................... 181

Figure 5.3: Notched IZOD Cross-Section SEM Morphology of A) PHBV, B) PHBV/DDGS

85/15 and C) PHBV/DDGS 75/25 Impact Samples After Break. .............................................. 184

Figure 5.4: Cellulose and PHBV/DDGS Biocomposites A) CO2 Evolution and B) Overall

Biodegradation. ........................................................................................................................... 187

xiii

LIST OF ABBREVIATIONS

Abbreviation Full Name

4HB

Bio-PE

Bio-PET

CA

CAB

CAP

CE

DCOI

DDGS

DMA

DS

DSC

HB

HDPE

HDT

HO

HP

HV

Hx

LDPE

Misc

PBAT

PBS

PCL

pCA

4-hydroxybutyrate

Bio-based poly(ethylene)

Bio-based poly(ethylene terephthalate)

Cellulose acetate

Cellulose acetate butyrate

Cellulose acetate propionate

Chain extender

4,5-dichloro-2-n-octyl-4-isothiazolin-3-one

Distillers dried grains with solubles

Dynamic mechanical analysis

Degree of substitution

Differential scanning calorimetry

Hydroxybutyrate

High density poly(ethylene)

Heat deflection temperature

Hydroxyoctanoate

Hydroxypropionate

Hydroxyvalerate

Hydroxyhexanoate

Low density poly(ethylene)

Miscanthus

Poly(butylene adipate terephthalate)

Poly(butylene succinate)

Poly(ε-caprolactone)

Plasticized cellulose acetate

xiv

PE

PEA

PES

PET

PH4B

PHA

PHB

PHBHx

PHBV

PHBO

PHBP

PLA

PP

PPP

PS

PTMA

PTT

PVOH

ROP

SA-GMA

SEM

Tc

Tg

TEC

TGA

TPS

Xc

Poly(ethylene)

Poly(ethylene adipate)

Poly(ethylene succinate)

Poly(ethylene terephthalate)

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

Poly(hydroxyalkanoate)

Poly(3-hydroxybutyrate)

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

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Poly(3-hydroxybutyrate-co-3-hydroxyoctanoate)

Poly(3-hydroxybutyrate-co-hydroxypropionate)

Poly(lactic acid)

Poly(propylene)

Poly(p-phenylene)

Poly(styrene)

Poly(2,2,6,6-tetramethyl-piperidenyloxyl-4-yl methacrylate)

Poly(trimethylene terephthalate)

Poly(vinyl alcohol)

Ring opening polymerization

Poly(styrene-acrylic-co-glycidyl methacrylate)

Scanning electron microscopy

Crystallization temperature

Glass transition temperature

Triethyl citrate

Thermogravimetric analysis

Thermoplastic starch

Degree of Crystallinity

»

xv

LIST OF PUBLICATIONS

Peer Reviewed journal Articles

[Chapter 2] Kjeld W. Meereboer, M. Misra, A. K. Mohanty. Review of Recent Advances on

Biodegradability of Polyhydroxyalkanoate (PHA) Bioplastics and their Green Composites. To be

submitted to Green Chemistry, April 2020.

[Chapter 3] Kjeld W. Meereboer, A. K. Pal, M. Misra, A. K. Mohanty. Sustainable

PHBV/Cellulose Acetate Blends: Effect of a Chain Extender and Plasticizer. ACS Omega,

(Accepted March 2020).

[Chapter 4 & 5] Kjeld W. Meereboer, A. K. Pal, E. O. Cisneros-López, M. Misra. A. K. Mohanty.

Marine Biodegradation Behaviour of PHAs and Natural Filler-based Biocomposites. To be

communicated to Scientific Reports, May 2020.

1

Chapter 1: Introduction

1.1 Structure of Thesis and Connecting Statements

The thesis structure is based on the study of bio-based and biodegradable polymers and their

blends and biocomposites. The current chapter outlines the research problem of the project, the

objectives and hypothesis of the research problem, as well as the significance of completing the

project objectives.

Chapter 1 identifies the knowledge gap of bio-based and biodegradable polymers that limit

their use in industry. Chapter 2 gives a detailed literature review of poly(hydroxyalkanoa te)s

(PHAs), PHA blends and its biocomposites; production, properties, and biodegradation in all

natural and synthetic environments following ASTM standards. Chapter 3-5 give a very detailed

review and study of PHA blends and biocomposites development and their thermal, mechanica l,

morphological and biodegradable properties.

The literature review of Chapter 2 indicates PHA biodegradation is well studied in all aerobic

and anaerobic environments. However, major limitations are the mechanical properties which can

only be improved by blending with non-bio-based or non-biodegradable polymers. Therefore, the

first study in Chapter 3 focuses on the development of a potential biodegradable blend based on

poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and cellulose acetate (CA), and their

mechanical, thermal and morphological properties. This study was completed to ascertain if PHBV

and plasticized CA were miscible and could produce a flexible bio-based polymer with

biodegradable behaviour.

Chapter 4 investigates the development of PHBV and Miscanthus fibre biocomposites and

their marine biodegradation. Miscanthus is an agricultural residue of low-cost that can significantly

reduce the PHAs. The effects of 15 and 25% Miscanthus loadings in PHBV on the mechanica l,

thermal and morphological properties were investigated. Furthermore, the marine biodegradable

properties were investigated utilizing the new ASTM D7991-15 marine biodegradation standard.

Marine biodegradation was completed to supplement the PHBV-based biocomposite knowledge

2

gap found in literature with the hopes to determine if natural fibres improve the marine

biodegradation performance.

Chapter 5 studies the effect of a proteinaceous natural filler on the properties of PHBV.

Distillers dried grains with solubles (DDGS) were washed and incorporated into PHBV to improve

the marine biodegradation rate further than Miscanthus fibre at 15 and 25% loadings. The

mechanical, thermal and morphological properties were investigated, and marine biodegradation

was studied as per ASTM standard.

Chapter 6 summarizes the main findings of each study and the potential future works and

avenues of interest which could be completed.

1.2 Problem Statement

Environmental concern around the petroleum plastic industry greenhouse gas emissions has

led to the development of sustainable bio-based plastics. However, the impact of mismanaged

plastic waste remains unresolved and is projected to grow, impacting all types of aquatic life in

the world’s oceans. This project is intended to resolve the potential plastic waste accumulation in

marine environments by working with biodegradable polymers. PHAs are a unique polymer family

that show biodegradable behaviour in “all-natural” environments such as soil and marine water.

Furthermore, being bio-based its impact on greenhouse gas emissions is significantly lower. As

the shift to more sustainable approach to plastic development and use becomes apparent, PHAs

potential become more important. However, PHAs have poor mechanical properties on top of a

significantly higher price compared to other bio-based polymers.

Investigation of a biodegradable blend system containing PHBV and CA was completed.

CA is a low-cost biopolymer that can significantly reduce the cost of PHBV, and when plasticized

it has desirable flexibility. Literature indicates cellulose acetate derivatives may be miscible with

PHBV, thus it is the directive of this study to determine the miscibility and mechanica l

performance of PHBV and CA blends. CA is also known to be compostable, but not marine

biodegradable. The second facet of this research project was to take advantage of the marine

biodegradable properties of PHBV, by incorporating agricultural residues. Miscanthus fibre and

3

distillers dried grains with solubles (DDGS) were used as fillers in virgin PHBV and studied under

the new ASTM D7991-15 marine biodegradation standard. These natural fillers are low-cost and

may improve the marine biodegradation without negatively impacting the high stiffness PHBV is

known for. The PHBV-based biocomposites should improve the marine biodegradable properties

of PHBV.

1.3 Objectives and Hypothesis

1.3.1 Objective 1: PHBV Blending

Investigate the miscibility, thermal, mechanical and morphological properties of PHBV and CA

based blends.

1.3.2 Hypothesis 1

PHBV and plasticized cellulose acetate blend will be used to optimize the flexibility.

Improve the tensile elongation of PHBV based blends.

Investigate the miscibility through analysis of glass transition temperatures of PHBV and

CA blends.

Develop a flexible material with good impact and elongation properties.

1.3.3 Objective 2: Biodegradation of PHBV/Miscanthus Biocomposites

Develop PHBV-based Miscanthus biocomposite to improve the biodegradable properties.

1.3.4 Hypothesis 2

Miscanthus fibres will be incorporated into PHBV at 15 and 25% loadings.

PHBV/Miscanthus biocomposites will have an improved marine biodegradation rate

relative to PHBV.

4

Mechanical properties of PHBV/Miscanthus biocomposites will increase

Miscanthus fibres will reduce the crystallinity of PHBV fraction in biocomposites.

1.3.5 Objective 3: Biodegradation of PHBV/DDGS Biocomposites

Investigate the effect of proteinaceous DDGS in PHBV mechanical, thermal and biodegradable

properties.

1.3.6 Hypothesis 3

DDGS will be incorporated into PHBV at 15 and 25% loadings.

PHBV/DDGS biocomposites will have an improved marine biodegradation rate relative to

PHBV and PHBV/Miscanthus biocomposites.

Mechanical properties of PHBV will be minimally impacted by DDGS inclusion.

1.4 Significance

The development and investigation of these PHBV blends and biocomposites will identify key

advantages of biodegradable polymers over petroleum-based non-biodegradable polymers.

Plasticized CA can improve the flexibility of PHBV for applications of single use plastics

in industry.

Incorporation of Miscanthus fibre into PHBV will improve the mechanical performance

for rigid plastic applications.

Marine biodegradation of PHBV and PHBV-based biocomposites will be investigated

under ASTM D7991-15 for the first time with a comparable method.

Incorporation of natural fibres and fillers or biopolymers can add value to the agricultura l

residue industry and the corn ethanol production industry while also reducing the cost of

PHAs.

5

6

Chapter 2: Literature Review

Abstract

Detrimental impact of single-use plastic in the environment is daily news across the globe. Single

use plastic packaging materials and other plastic waste originating from petroleum-based sources

are continuously building up in landfills and leaching into the environment. Using recycling as a

solution has limitations due to mixed materials used in one product, contamination and degraded

performance properties of polymers when reused. Managing plastic waste remains an urgent crisis

in the environment and switching to biodegradable plastics can help mitigate some of these issues.

This review will summarize the recent advances and opportunities to utilize

Polyhydroxyalkanoates (PHAs) as a biodegradable substitute in some applications where non-

biodegradable and petroleum-based plastics are currently used. PHAs are a well-known family of

bacterial-based biodegradable plastic. As such, they offer a potential to approach carbon neutrality

and support a more sustainable industry. PHAs such as poly(3-hydroxybutyrate) (PHB) and

poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) show biodegradable behaviour in all

aerobic and anaerobic environments defined by ASTM standards, and can be used to make

completely compostable and soil and marine biodegradable products – a strong positive compared

to the negativity associated with the landfilling of plastics. However, PHAs are relative ly

expensive compared to petroleum-based alternatives of comparable properties. To reduce the cost,

PHAs can be used in biocomposite materials, where incorporation of bio-based agro-residues,

fibres and fillers can lower costs while maintaining the performance in certain industr ia l

applications.

Organic fillers and fibres can improve the properties of polymers, however, their effect on the

marine biodegradable properties of the composite matrix remains an unexplored area. Generally,

fibres and fillers from the biosphere are considered biodegradable if containing cellulose and

cellulosic material. When used in biocomposites with PHAs, they improve PHA biodegradation

rates in all environments. In addition to cellulose, other bio-based fillers and fibres such as proteins

(i.e. distillers dried grains with solubles, soy meal), and starch have been reported to significantly

7

improve the soil and marine biodegradability rate compared to other fibres and fillers. Another

component that affects biodegradability are chemical additives (i.e. chain extenders) and

compatibilizers (i.e. maleic anhydride etc.) that are added to plastics to optimize the service life

properties, but are reported to inhibit the biodegradation start, rate and extent by impacting the

hydrophilicity of the polymer and enzyme activity. The multitude of possible combinations of

polymers and fillers and fibres, and their effect in the biodegradation of PHA-based biocomposites

is a largely unexplored frontier. The potential benefits of PHA-based biocomposites make a strong

case for further research into this area.

8

2.1 Introduction

A seismic shift in economic objectives triggered by the growing and overwhelming evidence from

industry, suggests that the projected cumulative growth of primary plastic waste produced by 2050

will exceed 25 billion metric tons 1. Combined with the shift towards sustainability using non

petroleum based plastics, the production of bio-based/non-biodegradable and biodegradable

plastics projected from 2020 to 2023 2 is expected to grow 13 % per annum. Leading plastic

packaging producers are moving towards a goal of 100% recycled, biodegradable or re-useable

plastic in their products by 2025 3. This shift towards a sustainable economy has occurred in the

recent decade, such that, between 2010 and 2017, bio-based poly(ethylene) (Bio-PE) bio-based

poly(ethylene terephthalate) (Bio-PET), poly(lactic acid) (PLA) and poly(hydroxyalkanoa te)s

(PHAs) have seen production capacity growths of approximately 22%, 10,000% 300% and 41 %

respectively 4. Replacement of petroleum-based plastics with bio-based alternatives is a more

sustainable pathway to plastic production due to their lower associated carbon emissions from

petroleum extraction and refinement 5.

2.1.1 Polymer Pollution in the Environment

Plastic pollution occurs in two fronts, during production (carbon emissions) and their disposal

(contaminants and physical hazards) which impacts both the environment and the ecosystem.

Replacing petroleum-based polymers with bio-based polymers is a potential solution that produces

significantly lower carbon emissions and energy production requirements 6. Figure 2.1 illustrates

the reduced impact of bio-based/biodegradable polymers on the environment relative to some

commercial petroleum-based polymers. However, regardless of their production method most

plastic waste after their service life ends up being incinerated, landfilled, littered or recycled,

resulting in carbon or methane emissions over time. It is the disposal after the service life of

biodegradable polymers that further benefit the environment compared to non-biodegradab le

petroleum-based polymers. Non-biodegradable polymers can leak contaminants or additives into

soil and waterways, and physically obstruct animal digestive systems 7. Several industries can thus

benefit from this as there is increasing carbon footprint reduction measurements and reporting

required due to increased societal pressure on industry to be more environmentally responsible.

9

HDPE

LDPE

Nylon

6PET

PS

PVOH

PCL

TPS

PLAPH

A0

30

60

90

120

150

Energ

y R

equirem

ent

(MJ/k

g)

Petroleum-based Bio-based

0

2

4

6

8

10

Glo

bal W

arm

ing (

kg C

O2 e

q/k

g)

Figure 2.1: Production Energy and CO2 Emissions of Petroleum-based and Bio-based Polymers 6,8.

2.1.1.1 Polymer Waste Disposal Streams

The polymer waste disposal can generally be divvied up into four separate outlets (Figure 2.2),

landfill, leakage, incineration and recycling which each have their own drawbacks 9:

i. recycling produces some losses and material degradation;

ii. incineration produces energy at the cost of material and pollution;

iii. leakage results in environmental hazards which can harm the surrounding environment;

iv. and landfills result in uncontrolled degradation that can severely harm the environment from methane production.

A new subset of controlled or managed degradation and disintegration is now being developed

with the implementation of compostable polymers in industry, following ASTM standards. The

10

end-of-life is value-added usable compost, which could evolve into a multi-billion dollar in and of

itself generating employment for thousands.

Figure 2.2: Predominant Current Polymer Waste Disposal Streams 9.

2.1.1.1.1 Recycling

Recycling plastics is an option to reduce the overall plastic waste produced, however, recycled

plastics suffer from reduced mechanical performance. These can be overcome by physically

treating (annealing) polymers to increase modulus and strength, chemical stabilizers, blending with

other recycled plastics or blending recycled plastics with other polymers as a valorisation method

10 . Irrespective of this, recycling generates waste during or at the end of the multi-recycling cycle,

with the material being too degraded to use. After many years of implementing recycling, still only

a small percentage of plastic in the US is actually recycled (<10%), compared to non-plastic

recycling (25-65%), in 2017 11. Therefore, an alternative method of plastic waste disposal is

11

required when the service life has ended. Possible alternatives include composting, or diversion to

alternative energy production (incineration).

2.1.1.1.2 Landfill

Landfills are suitable for storage of plastic waste, in comparison to other environments, due to the

ease of human intervention. However, landfills go through uncontrolled degradation, releasing

greenhouse gases into the environment. Landfills can be subdivided into several types, based on

age and the type of material waste. In consideration of municipal solid wastes, there are old

landfills and modern landfills. Old landfills have no control of pollution migration and no gas

capturing technology in place. Modern landfills are designed to capture the methane produced, for

energy generation 12. However, in less developed regions, landfills do not have these measures 13

and the off-gases can migrate into the surrounding area 14. Commodity plastics are not generally

landfilled, and, unlike organic material, do not degrade into methane. But this poses another

problem because it remains in the landfill indefinitely under anaerobic conditions. Furthermore, if

mismanaged waste results in biodegradable polymers being landfilled, the methane generation can

make up to 50% of the total gas release 15. Another environmental factor is the proper soil coverage

after closing the landfill, to prevent plastic waste to be scattered and dispersed into the environment

16.

2.1.1.1.3 Leakage (Litter)

Plastic waste in the environment is grouped into two sources, marine-based and land-based,

however the relative quantity remains unclear 17. Examples of ocean-based waste that likely

remains in the ocean today would be lost cargo and plastic pellets from shipping services 18, or just

general shipping waste pollution. Other pathways involve migration of plastics from land (i.e.

litter, landfills) to the ocean by environmental elements 18. Land-based plastic litter can be

accidental through environmental elements or intentional, usually by inadequate waste disposal

facilities during events or in the public spaces 19. The plastic litter can also originate from landfil ls,

sewage systems and industrial processes which degrade overtime and accumulate in the soil 17.

Regardless of the sources, plastics containing additives, such as plasticizers, UV stabilizers

12

contaminate the soil and marine environment overtime and impact animal and cellular organ

function 7.

With migration of plastics in the environment it is expected plastic can be found in a number of

oceans (surface and sea floor), shorelines and lakes across the globe 17. Plastic ending up in the

ocean is of concern due to their movement and the difficulty of human intervention. Plastic debris

in the ocean can have concentrations up to 580,000 pieces/km2 20. This plastic has been found in

the Pacific, Atlantic, and Indian oceans in the past two decades, especially in the North Atlantic

gyre and the North Pacific subtropical gyre where garbage patches have seen significant growth

21–23, and it is predicted 99% of all species of seabirds will ingest plastic by 2050 20. Furthermore,

this plastic lingers in the environment due to its durability if not exposed to microorganisms or UV

radiation 17. This is exacerbated by the protective coatings applied to polymers to ensure the

properties are not damaged by UV exposure during their service life 24. This plastic residing in the

environment can’t be recycled due to contamination or poor residual mechanical and thermal

properties. Therefore, biodegradable plastic holds significant importance in combatting

mismanaged waste which is expected to double by 2025 25.

2.1.1.1.4 Compost

Composting is a subset of biodegradation, such that not all compostable materials are

biodegradable in other environments such as marine, soil, landfills etc. Furthermore, only a small

subset of plastics can be composted, and do not include the commodity recycled ones. Composting

can be divided into home composting and industrial composting, with the main difference being

the controlled conditions in the industrial composting (~58 °C, 50-55% solids etc). In most

climates, home composting is slower than industrial composting, but is suitable for composting

organic material due to their short degradation period 26. Composting of bioplastics is mainly

limited to industrial composting operations and not recommended for home composting 27.

Industrial composting is designed for large amounts of organic waste, has a high turnover and

produces compost suitable for soil remediation 28.

13

Compostable plastics are predominantly thermoplastic starch (TPS), PLA and poly(butylene

adipate terephthalate) (PBAT), making up 83% of the biodegradable plastic produced in 2018.

Poly(butylene succinate) (PBS), another biodegradable plastic, has also experienced growing

global production capacity due to the significantly lower production cost compared to other

biodegradable polymers (i.e. PHAs) 29. Biodegradability of these plastics vary, with some (i.e.

PLA) being less suitable for home composting due to the long duration 30, such that only industr ia l

composting conditions are suitable. Furthermore, bio-sourced plastics utilize renewable carbon,

compared to petroleum-based biodegradable polymers (PBAT). Given industrial and commercia l

composting is not always suitable for compostable plastics, an alternative bio-based biodegradable

polymer such as PHAs is well posed for commercial adoption use due to better biodegradable

properties in many types of environments 31.

Therefore, the use of bio-based and biodegradable plastics that can degrade in natural

environments (i.e. soil, ocean water etc.) as a global movement is important because it combats

both climate change and plastic pollution – both necessary for sustainable growth and lowering

the carbon footprint for positive environmental affect. An important factor to consider is how bio-

based biodegradable plastics differ from other plastics, both in terms of their production and at the

end of life? Any modifications on the biodegradation of these plastics, including fibre and filler

addition, blends and chemical additions must be considered under a comparable standard in all

environmental conditions.

2.2 Bacterial-Polyesters

Among bio-based can be subdivided into three types, plant-based (i.e. starch, cellulose derivatives

and natural rubbers), polymerized bio-monomers (i.e. PLA, polyimides, polyurethanes,

poly(butylene succinate) (PBS), bio-PE etc.) and extracted bio-polymers (PHAs) 32,33. Further

subdivision of polymerized bio-monomers exist with partially bio-based polymers such as nylon-

6,10, poly(trimethylene terephthalate) (PTT) and poly(p-phenylene) (PPP) 34,35. Bacterial

polyesters are a unique subset of bio-based polymers polymerized by microorganisms. PHAs for

example can be synthesized enzymatically in vivo by microorganisms as a true natural polyester

for intracellular storage 36, while PLA is produced through fermentation as lactic acid and

14

chemically polymerized 37. However, despite differing production methods, not all bio-based

polymers are biodegradable. Among bio-based polymers, only PLA and PHAs are completely bio-

based and biodegradable in some form. However, PLA is compostable, but not marine

biodegradable like PHAs, making it unsuitable to combat plastic waste leaking into the

environment 38. Moving towards bio-based biodegradable polymers allows for a more sustainab le

option by implementing a cradle-to-cradle approach 39, where the outputs of biodegradation

becomes the production inputs for the same polymer within a reasonable frame of time (Figure

2.3). For example, despite PE and bio-PE being petroleum based and bio-based respectively, their

main disposal stream is through either recycling or landfill. In addition, petroleum-based polymers

such as PBAT are biodegradable 29, but due to their production method, do not form a circular

cradle-to-cradle approach. PHAs, PLA and cellulosic material are all bio-based and biodegradable,

forming a complete carbon cycle, however, PLA is only compostable, not marine biodegradable

like PHAs, making it unsuitable to combat the leakage of plastic into the environment 38. While

biodegradable plastic can potentially be recycled in the general waste stream, the resulting

properties of the recycled plastic is significantly worse and they are more suited for short life cycle s

40.

15

Figure 2.3: Cradle-to-Cradle and Cradle-to-Grave Approach of Biodegradable and Non-Biodegradable Plastic.

2.2.1 PHAs

PHAs are aliphatic polyesters well known for their biodegradable properties and their bacterially

based production methods. Over 91 different polyhydroxyalkanoic acid constituents that make up

PHAs have been recorded and the number is continuously growing 41. Based on the potential

combination of monomer units, an uncountable number of PHA copolymers can be formed. PHA

being biodegradable in various environments are an attractive option to replace current single use

plastics 11 or plastics that are unsuitable for re-entry into the manufacturing sector due to their poor

quality 42. With their biodegradable properties, PHA form a closed loop cycle from cradle-to-cradle

16

(Figure 2.3) that minimizes the impact on the environment 43. However, the functionality and

production methods are dependent on the type of PHA.

2.2.1.1 Classes of PHAs

The main subsets of PHAs can be categorized by their chain length: (i) short chain length of 3-5

carbons; (ii) medium chain length of 6-14 carbons; and (iii) long chain length of 15+ carbon atoms.

Among short and medium chain length PHAs are unique types with double bonds, when produced

from unsaturated fatty acids 44. The most well-known PHAs are poly(3-hydroxybutyrate) (PHB)

and poly(3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), both are the short chain PHAs and

represent the most basic forms commercially available. Other currently available types of PHAs

used in biodegradation studies are poly(3-hydroxybutyrate-co-4-hydroxybutyrate (PH4B), poly(3-

hydroxybutyrate-co-3-hydroxyhexanoate (PHBHx), and poly(3-hydroxybutyrate-co-3-

hydroxyoctanoate (PHBO). The functional components are illustrated in Figure 2.4. PLA has been

previously considered part of the PHA family 41, however, the polymer production is significantly

different compared to PHAs.

Figure 2.4: a) 3-hydroxypropionate (HP), b) 3-hydroxybutyrate (HB), c) 3-hydroxyvalerate (HV), d) 4-

hydroxybutyrate (4HB), e) 3-hydroxyhexanoate (Hx), f) 3-hydroxyoctonaote (HO) chemical structures.

17

2.2.1.2 Production of PHAs

The synthesis of PHAs is important in biodegradation as the metabolic pathways are related to bio-

assimilation. PHAs are usually produced by recombinant Escherichia coli for commercial use 45

but can be produced by a number of other microorganisms (i.e. Aeromonas, Azotobacter,

Cupriavidus, Clostridium, Methylobacterium, Ralstonia, Pseudomonas, Syntrophomonas etc.)

15,44,46. Pseudomonas is the only reported species to produce long carbon chain PHAs 47.

The bacterial production of PHAs begins under growth limiting conditions because it functions as

an energy storage molecule for microorganisms 48, leading to accumulation in the cell walls of

bacteria and archaea 49,50. HB monomers are then bacterially polymerized by PHB synthase of

acetoacetyl-CoA 51, however chemical pathways do exist such as ring opening polymeriza t ion

(ROP) of β-butyrolactone 52. PHBV is manufactured under similar conditions but with the presence

of propionic acid. The copolymerization process is initiated enzymatically from acetoacetyl-CoA

and 3-ketovaleryl-CoA to form the HB and HV units, respectively 53,54. However, with additiona l

nutrients and energy costs making up more than 75% of the product cost 55, PHAs become a

relatively expensive product compared to commercial petro-based polymers of comparable

properties 56. For bacterial PHA production, several non-conventional carbon feed sources are

used, including mixed carbon sources 57, organic wastes 35, methane 58, peanut oil 59, soybean oil

60,61, palmitate oil 62, waste frying oil 63, margarine waste 64, and glycerol etc. 65. Despite these

advantages, the costs associated with the production of PHAs are still very high (comparative to

other bio-polymers) and the properties leave much to be desired. Furthermore, while petroleum-

based biodegradable plastics can be incorporated into PHAs, to reduce costs and mainta in

functionality, the sustainability of production becomes lower.

2.2.1.3 Applications of PHAs

Due to PHAs biocompatibility and biodegradability, they are considerably attractive for temporary

in vivo applications, and several have been developed for both PHB and PHBV where high

production cost is less significant. Among these applications are antimicrobial releasing sutures 66,

18

cellulose support films for gas transfusion 67, long-term drug release capsules 68, bone tissue fibres

for osteoblast growth 69, and other tissue scaffold applications such as neural regeneration 70.

Applications of PHAs outside of the biomedical industry are mainly in replacement of for single

use, disposable plastics, such as plastic tableware, food packaging, plant pots and organic waste

collection 71. PHAs also have potential for substitute poly(ethylene) (PE) in some film applications,

e.g. mulch film used to stop weed germination in agricultural farming. PHAs and blends of PHAs

with other biodegradable polymers show promise to fulfil the same role as PE with added benefit

of degrading during the season to reduce labour costs and farm waste at the end of the season 72,73.

In all cases, biodegradation is a critical factor that makes PHAs both marketable and defines its

applications outside of the biomedical industry. Currently, PHA use in single use plastics for the

food industry is limited, despite it showing potential for bottles, caps, blister packs etc.74, due to

odour issues that require additives to remove them 75. However, the biodegradation of the resulting

new blends with any additives remain to be fully explored.

2.2.2 Challenges of Biodegradable Plastics

Biodegradable plastics usually suffer from to limitations, poor mechanical properties (Figure 2.5),

and the inability to blend them with many other polymers without losing their biodegradable

functionality. The market value of biodegradable plastics is in their biodegradable performance

when disposed and still requires biodegradation testing to ensure no chemical interactions hinder

the overall biodegradation rate. The two main bio-based and biodegradable polymers, PLA and

PHB are both extremely brittle plastics relative to commodity and engineering plastics. Although

some PHA are more ductile as their chain length increases 76–78, the cost of PHAs are significantly

higher than conventional plastics and other bio-based biodegradable plastics 79. While

incorporation of petro-based biodegradable polymers is a potential solution to maintain the

biodegradable properties of PHAs, the resulting blends become less environmentally sustainab le

due to petroleum usage. For example, to make PLA more flexible, it is blended with PBAT and

marketed as Ecovio by BASF. Some studies have investigated blending PHAs with other bio-

based or biodegradable polymers. PLA was blended with PHB for improved ductility and thermal

19

stability 80, and poly(Ɛ-caprolactone) (PCL) was blended with PHBV for increased ductility 81.

However, the resulting blends suffer from reduced biodegradable performance or costs.

The commercial PHB and PHBV have low impact strength, high brittleness and poor flexibility,

making it unsuitable for many industrial and commercial applications compared to other

biodegradable or commercial polymers 82,83. Much success has been found in varying PHA

copolymer compositions, such as increasing the Hx content in PHBHx for improved flexibility

marketed as Nodax by Makena 84, or increasing the 4HB content to obtain elastomeric properties

85. However, with increasing complexity of PHA monomer units, the cost of production increases.

Natural fibres are an option to minimize the cost of PHAs while enhancing their best properties

such as modulus and biodegradation. However, the information about natural fibres, composition

and compatibilization techniques effects in biodegradation remains unclear.

20

Figure 2.5: Young's Modulus and Elongation at Break of Plastic Types. Reprinted (adapted) with permission

(Creative Commons) from Rastogi et al. (2015) on April 29, 2020 86.

2.3 What is Biodegradation?

Biodegradation is the degradation process involving microorganisms, and is widely accepted as

selective, and depends on several factors including the physical and chemical properties of

biopolymers. The biodegradation process is defined as a polymer degrading by biologica l

microorganisms into CO2, H2O, biomass and methane by composting, soil biodegradation, marine

biodegradation, or other biodegradation processes 56. It is also termed biotic degradation and can

be enhanced or started after some initial abiotic degradation processes occur such as mechanica l,

oxidative or hydrolytic degradation (Figure 2.6) which can increase the surface area of organism-

polymer interface 87,88. Thermal or oxidative degradation are non-selective, occurring to all

polymers, and introduce thermal or chemical stressors that scission the chains of polymers into

21

smaller units of oligomers, acids, alcohols, esters, and radicals 89,90. For example, PHAs are less

thermally stable than PLA and PCL due to the different thermal degradation mechanism of random

chain scission instead of unzipping depolymerization, and thermal degradation results in reduced

molecular weight along with the esters, and alcohol end groups 91. However, thermal energy can

also enhance most if not all other forms of degradation, through disproportionaltly increasing the

high energy collishions between reactants or enzymes and reactants. Catalytic degradation of

polyolefins results in gas, oil and waxes by clays, acids, zeolites, aluminium oxides, calcium oxides

etc. under high temperature 92. High temperatures are not desired for biodegradation of polymers,

due to the inactivity of microorganism enzymes.

The value of biodegradable polymers lies in rapid degradation under natural conditions (i.e. soil,

ocean water etc.) it can become the commercial attribute that reduces potential improperly

disposed of plastic waste 93. Multiple factors affect biodegradation besides the environment

conditions (i.e. temperature, moisture etc.); the polymer composition, molecular weight,

crystallinity, composition, chemical structure, reduction potential, hydrophilicity, breakdown

products etc. 94, but the extent of the affects from some of these factors remains unclear.

22

Figure 2.6: Basic Degradation Pathways of Polymers.

When recycling or incineration are unsuitable, biodegradable plastics offer an advantage that has

positive effects in environmental waste management. The process can be further enhanced by

utilizing bio-based biodegradable polymers, whereby the carbon source input of production,

biodegrades into CO2, which is then taken up to produce carbon source, thereby approaching

carbon neutrality.

Biodegradation is influenced by the susceptibility of the polymer carbon backbone to microbia l

attack 95. Degradation of any polymer can be divided into two types, surface erosion and bulk

erosion. The biotic (enzymatic) degradation is mainly at the surface. The reason is that enzymes

are relatively large particles and are unable to permeate the structure of polymers, in comparison

to smaller chemicals, free radicals etc. The abiotic degradation functions as both bulk and surface

degradation and is often used a pre-treatment to biodegradation. In general, bulk erosion is used

for breaking sample apart into smaller pieces (which enhances the rate of surface erosion) and for

molecular weight reduction. For example, poly(α-hydroxy-esters) samples must exceed a critical

minimum thickness (Lc) of 7.4 cm to undergo surface erosion and not bulk erosion (Figure 2.7) 96.

23

Figure 2.7: Bulk Erosion and Surface Erosion Effects on Polymers as Function of Time. Redrawn with

Permission (Lic. #4818250646220) from Van Dijkhuizen-Radersma et al. (2008) on April 29, 2020 97.

Enzymatic hydrolysis of polyesters is completed by lipases and esterases to break ester, carbonate,

amide and glycosidic bonds 98. Due to this nature, warmer temperatures (15-37 °C) and alkali

environments increase the rate of PHA and PLA degradation 99–101. Non-polyesters, considering

homochain polymers only, made from entirely carbon backbones are not readily biodegradable.

Microbes have difficulty enzymatically cleaving and degrading aliphatic homochain and

heterochain polymers without functional groups (i.e. esters, ethers etc.) such as PE, polycarbonate

(PC), polypropylene (PP), polystyrene (PS) and their derivatives with carbon backbones, and any

degradation observed is usually minimal 102–105. The degradation of polymers follows the

schematic of Figure 2.8, where biotic or enzyme catalysed reactions is usually the more effic ient

method. Following polymer degradation by biotic or abiotic methods, the products can be bio-

assimilated by microorganisms and used for other needs such as growth and cellular respiration.

24

In environmental conditions, biodegradation of PHA following standards is mainly competed

extracellularly by extracellular enzymes that cleave the polymer into small enough units to bio-

assimilate 106. During extracellular biodegradation, where the PHA is in a highly crystalline form

(mainly PHB) 35. However, enzyme-catalysed biodegradation can occur in cells of

microorganisms, and is referred to as intracellular biodegradation 107. Intracellular PHA is present

in an amorphous state with disordered conformation covered by protein and phospholipids, and is

degraded when no alternative carbon source is available.

Figure 2.8: Theoretical Biodegradation Pathway of Polymer Material.

25

2.3.1 Aerobic vs Anaerobic Biodegradation

Biodegradation of polymers can occur under aerobic or anaerobic conditions, leading to varied

products. Aerobic degradation (in the presence of oxygen) mainly utilizes the oxygen as a final

electron acceptor, while microorganisms that perform anaerobic degradation (in the absence of

oxygen) use CO2, nitrates, sulphates etc. as alternate electron acceptor to generate energy for the

cell functions 108–110.

Most biodegradable polymers show evidence of degradation in both aerobic and anaerobic

environments 111. For enzymatically degraded polymers, it is the temperature that usually affects

whether polymer scission occurs. For example, PLA requires a temperature equal or greater than

its Tg (~55 °C) to biodegrade effectively 112.

The basic chemical equation for aerobic biodegradation is the conversion of organic carbon into

CO2 by microorganisms, in the presence of oxygen (Equation 2.1). The carbon atom is generally

part of a complex structure, and in some cases the oxygen can be derived from the polymer itself,

such as from polyesters. During cellular respiration of a carbon source (i.e. glucose), the liberated

oxygen reacts with free hydrogen ions to produce water (Equation 2.2) 113.

𝐶 + 𝑂2 → 𝐶𝑂2

Equation 2.1

𝐻+ + 𝑂2 → 𝐻2𝑂

Equation 2.2

Anaerobic biodegradation on an industrial scale following standards is a significantly less studied

area compared to aerobic biodegradation, due to the environment control required for the study,

and it does not constitute the majority of natural environments where plastic waste could

biodegrade (soil and marine) The stoichiometric mass balance of anaerobic biodegradation of

plastics and natural fillers is defined by Equation 2.1, Equation 2.3 and Equation 2.4.

26

𝐶 + 2𝐻2 → 𝐶𝐻4

Equation 2.3

𝐶6𝐻12𝑂6 → 3𝐶𝑂2 + 3𝐶𝐻4

Equation 2.4

Under anaerobic conditions, biodegradation results in methane production and some CO 2 is

produced depending on the residual oxygen in the environment or the type of degraded material.

Two types of anaerobic environments exist on a large scale due to commercial actions, biogas

facilities and landfills. Biogas facilities deal with anaerobic digestion of organic and plastic

material, capturing the released methane for energy conversion 114. However, landfills are of

particular concern, because any uncontrolled biodegradation of organic and plastic material can

result in methane generation into the environment. In 2007, it was estimated only 10% of the

potential methane generated is captured in the United States 115, which has only increased to

approximately 20% by 2017 114. Methane is a 25 times more potent greenhouse gas compared to

CO2 over a 100-year period. Furthermore, in the waste sector, the largest contributors of methane

in the atmosphere are the landfills and solid waste treatment facilities which do not collect biogas,

indicating uncontrolled management of these greenhouse gases 116.

2.3.2 Misinterpretation of Oxo-degradable Plastics

However, another cast of biodegradable polymers has been defined, as oxo-degradable under

ASTM standards. Oxo-degradable plastics undergo abiotic degradation before undergoing either

aerobic or anaerobic biodegradation. There is significant concern about defining plastics as oxo-

degradable and interpreting it as biodegradable in the plastic community 117. Oxo-degradable is

the physical degradation of plastics into smaller units through oxidation, thermal or ultravio let

actions. Through these processes the molecular weight decreases, and potentially these monomer

units could be simpler to bio-assimilate. However, this is not always the case if the final units have

little to no functionality or a lack of carbon, nor is it a rapid process.

27

The claim of oxo-degradable has previously been termed as biodegradable by several literature

sources, but discrepancies are found in the pre-treatment which applies unrealistically high or

accelerated conditions (i.e. thermal, UV exposure etc.) in temperature and duration respectively.

These studies found 400-600 days are required to obtain at least 30% biodegradation for LDPE

films 118,119. ASTM D6954-18 indicates something like an oxo-degradability standard, but it must

be used with other biodegradations standards (i.e. soil etc.) in the presence of these abiotic

actuators.

There are three tiers to the oxidation and biodegradation standard 120 (Figure 2.9):

1) Abiotic degradation to a weight average molecular weight of 5000 or less. Products vary

depending on the polymer polarity and type of initiator. Oxidation of non-polar molecules gives

free radicals, UV or heat treatment gives hydrolysed molecules with functional groups.

2) Biotic degradation in environment of choice (stated compost, soil or accelerated landfil l

conditions). The products vary based on the standards followed.

3) Measure of toxic residue.

28

Figure 2.9: ASTM D6954-18 Oxidation and Biodegradation Standard Test Procedure 120.

There are some claims of oxo-degradable LDPE plastic packaging and UV-degradable plastic,

however it only physically degrades, and does not produce CO2 38. These types of packaging can’t

be deemed biodegradable in such cases as they physically degrade into microplastics, and

biodegradation is not achieved, which is of significant concern for the potential harm chemica ls

and polymers can cause on the wildlife both physically and chemically when ingested 121 and of

particular concern is the extremely small particles that can find their way into the water systems

and cause severe harm to ocean-life.

29

2.3.3 Aerobic Biodegradation Standards for Polymers and Their Limits

This review will use consider American Society of Testing Materials (ASTM) that defines three

main areas of aerobic biodegradation standards: soil biodegradation, composting, and marine

biodegradation, and three anaerobic biodegradations standards: sewage sludge biodegradation,

anaerobic digestion biodegradation, and accelerated landfill biodegradation.

This review does not include standards for unique conditions such as European ISO 14851 and

ISO 14852 which are used to Certify the OK Biodegradable Water designation in Austria 122.

Furthermore, this study does not include aerobic biodegradation standards withdrawn (not updated

within time limit), including the following: ASTM D5209-92 in 2004, ASTM D5271-02 in 2011,

and ASTM D6340-07 in 2016, which represent wastewater and sludge aerobic biodegradation,

due them being uncommon processes compared to the available aerobic biodegradation standards.

One major limitation of these withdrawn standards are the static test conditions, whereas the

natural environment is more susceptible to changes in conditions from climate, weather etc.

2.3.3.1 Soil Biodegradation

Research in soil biodegradation can be divided into two categories, those that indicate the degree

of biodegradation, and those that indicate the mass loss over the duration of the study. The latter

is more prevalent in literature due to its ease, however, based on the ASTM standards, it is not

enough to determine the degree of biodegradability of polymers by itself. Furthermore, it is the

duration of soil biodegradation that make complete studies following ASTM standards incredib ly

rare.

Soil biodegradation occurs when biodegradable material is exposed to soil microbiomes, close

enough to the surface to be in an aerobic environment. It does not follow the same level of

awareness as composting in society due to their being no implemented collection systems. ASTM

D5988-18 and its equivalent ISO 17556 for soil biodegradation only requires the initial conditions

of the soil to be reported (i.e. pH, moisture content, moisture holding capacity, ash content, carbon

to nitrogen, etc.)123. Variations in moisture can alter the degradation rate of hydrolytically driven

biological processes, dry conditions slow hydrolytic reactions, thus the biodegradation studies may

30

not be comparable. This test can also be complemented with mechanical properties and physical

degradation identified in ASTM G160-12, but there is no soil biodegradation ASTM labelling

standard that defines whether something can be claimed as soil biodegradable.

The temperature is limited to a range of 20-28 °C, not reflecting a holistic climate approach,

however, the soil must be collected from the surface of any natural environment, allowing for some

malleability in studies of natural environments. It is also important to note that the efficiency and

quantity of cellulose degraders vary based on the type of soil and location 124, which would be

further reflected in the polymer degraders diversity. Therefore, climates, region and the

temperatures of the soil and the preceding winters all affect the type and the quantity of

microorganisms in and around the soil.

2.3.3.2 Composting

The most well-known form of biodegradation is composting (ASTM D5338-15 and its equivalent

ISO 14855), mainly due to the labelling standard that can be used to define whether something is

compostable (ASTM D6400-19), in North America. Biodegradable material is exposed to a

mixture of decomposed material at higher temperatures than that found in soil biodegradation.

However, the compost standard is for industrial or commercial applications, such that the

environment is controlled or optimized in terms of parameters and initial conditions (i.e. moisture

content, carbon to nitrogen ratio (10-40) etc.), which forms a synthetic and stable environment.

The inoculum must have less than 70% ash, solids of 50-55% and a temperature fixed at 58 °C,

while the cellulose reference sample must be 70% degraded in 45 days less 125. For marketing

purposes, the labelling standard defines that 90% of the polymer must be physically degraded in

90 days, 90% of the polymer must be chemically degraded in 180 days and terrestrial safety

(impact upon plant growth) of the final compost must be within certain specifications, to be defined

as a compostable polymer 126. ISO 20200 is a composting disintegration study, which can be used

to supplement composting studies, however, it defines physical disintegrate-ability, and not

biodegradation.

31

Other composting standards available reflect more natural or home composting such as ASTM

D5929, which studies the composting of “organic” materials in aerobic conditions under

mesophilic conditions (25-45 °C), and has been applied to plastics 31. However, the standard

identifies the conditions to be maintained at 40 °C which is beneficial to biodegradation but not a

holistic approach to defining the actual reality of composting (ambient temperatures can vary

significantly from one geographical jurisdiction to another) and is not specifically designed for

plastic materials.

Three phases of composting exist 127:

1) The mesophilic phase: where easily degradable organic matter is broken down. The organic

acids produced reduce the pH to 5-5.5. With the rising heat from microorganism activity, the

proteins break down and pH increases to above 8.

2) The thermophilic phase: where the temperature rises above 40 °C and the degradation of waste

material improves. However, as the temperature continues to increase to 55-65 °C, the microbia l

activity reduces.

3) The “maturation phase: where the compost begins to cool with reduced microbial activity after

the readily available carbon sources have been consumed, hence “mature compost”. At this point

the mesophilic microorganisms take over and degrade the residual carbon sources over a long slow

process.

The three phases of composting are not well reflected in the testing procedures of ASTM D5338-

15, which stipulates a stable temperature. Furthermore, education of the end-consumer is needed

to impart a better understanding to avoid the improper disposal of compostable material (which

can be detrimental to the environment to the same extent as disposal of non-biodegradable plastics)

and allay misperceptions of what compostable means.

ASTM D6400-19 defines that something can be labelled compostable if it meets 3 tiers:

i) 1st Tier: 90% physically degraded to a particle size <2 mm in 90 days.

ii) 2nd Tier: 90% converted to CO2 within 180 days.

32

iii) 3rd Tier: no toxic residue in compost, and at least 90% germination rate and biomass of

plants grown in compost compared to blank.

2.3.3.3 Marine Biodegradation

Marine biodegradation is defined by three standards currently in use: 1) ASTM D6691-17, which

studies floating plastics at a temperature of 30 °C; 2) ASTM D7473-12, which evaluates plastics

buried in the sediment underwater by weight attrition only; and 3) ASTM D7991-15 which

requires plastics in a combination of water and sediment, with the option of light imitating day

light and a temperature of 15-25 ±2 °C. Figure 2.10 illustrates the limitations of each marine

biodegradation standard. ASTM D7473-12 is specifically indicated to be a supplementa l

assessment to ASTM D6691-17 which identifies their physical degradation and not

biodegradability, but it is useful to know if waterlogged plastics that have sunk will still physically

degrade. ASTM D7991-15 can replicate two locations of plastics, and like the other marine

biodegradable standards, the initial conditions need only be reported. However, ASTM D7991-15

only applies in the tidal zone, indicating plastics are close to the coast and the surface water 128.

A major limitation of marine degradation is no standard defines testing methods for polymers in

deep waters where temperatures are well below the optimal growth conditions for bacteria 129, and

pressure inhibits the rate of biodegradation and bio-assimilation of PHAs by microorganisms 130.

Furthermore, near the ocean floor, water movement is minimal, and anoxic (oxygen absence)

conditions can be common near the bottom sediment 131. Although ocean sediment is reported to

have a greater microorganism consortium that can degrade PHAs 132, it may not be enough to

mitigate these unfavourable conditions. Furthermore, water absorption is positively correlated to

temperature 133, which can detrimentally affect biodegradation in cooler climates and conditions.

Considering marine biodegradation will vary across the globe it is understandable that there must

be freedom in study. Several limitations have also been identified for biodegradation in aquatic

environments, such as differences between lab and real world conditions, benthic environments,

toxicity tests etc. 134.

33

Figure 2.10: Marine Biodegradation and Degradation ASTM standards.

To evaluate and claim marine biodegradability is difficult due to the lack of standards identifying

a threshold. ASTM D7081-05 did indicate a threshold for biodegradation to identify if polymers

were marine biodegradable (30% CO2 theoretical evolution and 70% physically degraded below 2

mm in 180 days). However this could only be applied on polymers tested using ASTM D6691-17,

and the standard has since been withdrawn 135. “OK Biodegradable Marine” designation is a valid

claim that can be made with 90% biodegradation achieved relative to cellulose or absolute, in

addition to physical degradation to a particle size below 2 mm in 180 days, and is supplementary

to ASTM D6691 136.

2.3.4 Anaerobic Biodegradation Standards for Polymers and Their Limits

Three main anaerobic studies exist, defining varying ratios of total solids seen in wastewater

treatment, landfills and anaerobic digesters. During anaerobic studies, since biogas is measured,

dissolved CO2 in water is not directly measured and must be accounted for afterwards which is not

done so in some older studies 137.

2.3.4.1 Sewage Sludge Biodegradation

Sewage sludge biodegradation is a highly active anaerobic study available in many developed

areas geographically. Defined by ASTM D5210-92, it is more representative of anaerobic water

34

biodegradation due to inoculum of 1-2% w/v. total organic solids. Such studies are reflected in

wastewater treatment plants available in many developed areas geographically. Test samples can

be in nearly any form (powder, fragments, pieces etc.) representing plastic material that has entered

the wastewater treatment facilities and are maintained at a temperature of 35 ±2 °C in dark

conditions. ASTM D5210-92 bears resemblance to ISO 14853.

As of the current date of this review, the standard has since been withdrawn for not being updated

in a timely manner but is still used in some studies. ISO 13975 is also similar to ASTM D5210-

92, for its inoculum type, however, the temperature can be either 35 or 55 °C 134.

2.3.4.2 Anaerobic Digestion Biodegradation

Anaerobic digestion is a static batch fermentation with 20% solids and can be considered a

synthetic method of biodegradation. ASTM D5511-18 and its equivalent ISO 15985 are studied at

either thermophilic (52 °C) or mesophilic (37 °C) conditions for a short period of 30 days. Since

there is large variation, the study is mainly to determine if the option is viable for polymers but

isn’t readily used in industry as a method of plastic disposal for energy recovery. The application

is considerably limited due to plastic incineration being faster and more cost-effective as a form

of energy production 138.

2.3.4.3 Accelerated Landfill Biodegradation

Landfill biodegradation can be considered the most undesirable form of biodegradation, due to

uninhibited and uncontrolled methane production under anaerobic conditions that is freely released

into the environment. Landfill biodegradation involves a sludge gestate (high organic content

material) solid content of 35% or greater, indicating a reduced amount of available water compared

to other biodegradation standards for biological activities. Two types of landfill biodegradation

standards exist, ASTM D5526-18, which is completed at 35 ±2 °C, and ASTM D7475-20 which

can be completed in aerobic and anaerobic conditions. ASTM D5526-18 has a sludge gestate total

solid content of 35, 45 and 60% and the extent of cellulose biodegradation must be above 70% in

30 days. ASTM D7475-20 has two tiers:

35

1) Biodegradation with household solid wastes of 50% total solids at 30 °C for 4 weeks.

2) Static digestion with a sludge gestate total solid content of 35, 45 or 60% and a temperature of

35 °C.

Landfills are generally packed densely to conserve space; therefore, biodegradation may be limited

due to lack of water or available CO2 or oxygen to properly degrade over time, in addition to the

lack of exposure to other degradation mechanisms due to the depth. The ideal scenario is

mismanaged biodegradable plastics end up in the appropriate stream such that it can aerobically

degrade instead of anaerobically, resulting in methane release in the environment. Among types

of PHA biodegradation, it is the least researched due to the limited studies found at this time.

2.4 PHA Biodegradation

As a biodegradable polymer, PHA has a few significant advantages for applications: (i) slow

release of chemicals such as fertilizers or pesticides in agriculture; (ii) photoactivation to induce

pollutant oxidation; (iii) leaving no residue behind within a short period of time 139; and (iv)

biodegradation pathways are similar to starch (about 90% of microorganisms that degrade starch

can also degrade a short chain PHAs 140). However, starch degraders can usually only bio-

assimilate (take in and utilize) biodegradation products and not perform complete biodegradation

with large molecular weight polymers or more complex medium to long chain PHAs.

Biodegradation has two types of microorganisms, those that physically degrade PHAs, and those

that feed off the by-products of the degradation (butyric acid, valeric acid etc.).

In this review, biodegradation studies completed in-vitro where samples are examined for

degradation and analysed based on surface diameter of degraded polymer, is not critica lly

evaluated. Studies following ASTM standards, modified ASTM standards, natural environments

and those in controlled environments (i.e. laboratory environments), with physical samples (i.e.

powder, films etc.), that show the extent of degradation and/or bio-assimilation are detailed

critically.

36

2.4.1 PHA Biodegrading Microorganisms

Holistically, short and medium length PHAs are biodegraded by a number of bacteria, includ ing

members of the genera: Actinomyces, Alcaligenes, Arthrobacter, Aspergillus, Bacillus,

Clostridium, Comamonas, Corynebacterium, Enterobacter, Gracilibacillus, Klebsiella,

Micrococcus, Mycobacterium, Nocardia, Pimelobacter, Planococcus, Pseudomonas,

Pseudoalteromonas, Staphylococcus, Streptomyces, Variovorax 129,141–149. The dominant PHA

destructors in aerobic and anaerobic environments have been reported by the members in the

bacterial genera of Variovorax, Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus,

Burkholderia, Cupriavidus, Mycobacterium and Streptomyces 143,150. Fungi can also degrade PHA

and are reported to be even more effective PHA degraders compared to bacteria 151. Known PHA

degrading fungi are in the division Ascomycota, Basidiomycetes, Deuteromycetes, Zygomycotina

in aerobic and anaerobic environments 152,153. Thus, it is reasonable to define PHAs as readily

biodegradable in most anaerobic and aerobic environments 10.

Under favourable conditions, biodegradation follows the ideal bacterial growth curve with a lag,

exponential, stationary and death phase, where the biodegradation plateaus between the end of the

stationary phase and the start of the death phase 154. However, if conditions are unfavourable, or

fluctuate throughout biodegradation, the rates can vary significantly from the theoretical model

due to this complex association between biodegradation and bio-assimilation of the products.

2.4.2 Extracellular PHA Biodegradation

The extracellular biodegradation of PHA is mediated by protein lipases and hydrolases. The

process follows the general step-wise approach of polymer breakdown into shorter chain polymers

by hydrolytic depolymerases in the presence of water, followed by further conversion of PHAs

into trimer and dimer units, which are then processed by lipases and hydrolases 155. The

extracellular PHA depolymerases are most studied; their protein structure consist of three main

domains: (i) a binding domain responsible for surface absorption and disruption of the polymer

structure; (ii) a linker domain that links the binding domain to the catalytic domain; and (iii) a

catalytic domain that cleaves the PHA and any available dimers/trimers in two (Figure 2.11).

37

Enzymatic hydrolysis of PHAs is a two-step process;

1) Adsorption of enzymes upon the surface of the polymer and active sites.

2) Enzymatic induced hydrolytic cleavage of PHA bonds which is induced by the hydrophobic

domain binding site and the catalytic site respectively 142,156.

The hydrolysis process is not very specific and releases oligomers of various sizes into the

surrounding medium 157,158. There is observed preference of hydrolytic enzymes toward

amorphous surface crystals, with less crystalline polymers and co-polymers being targeted more

readily due to their less ordered structure being more specially accessible to the enzymatic action

156,159. The degradation products vary depending on the type of PHA. In PHB, 3-hydroxybutyr ic

acid is produced, while PHBV products are 3-hydroxybutyric acid and 3-hydroxyvaleric acid 160.

These, acids are then taken into the cell to be metabolized into other compounds or more PHAs

51,161. Furthermore, the type of PHA impacts the way biodegradation proceeds effectively.

38

Figure 2.11: Single PHB Crystal Enzymatic Degradation by PHB Depolymerase. Reprinted with permission

(Lic. #4786571333526) from Iwata et al. (1999) on March 12, 2020 162.

2.4.3 PHA Attributes that Affect Biodegradation

Efficiency of PHA biodegradation is closely coupled with the physical and chemical attributes of

the polymer type. Degradations rates depend on the: (i) crystallinity; (ii) copolymers; and (iii)

copolymeric structure. In PHBV, the introduction of 3-hydroxyvalerate has a greater amorphous

region, which is more susceptible to enzymatic attack 52,163, due to eased water penetration 93,

absorption 164, and susceptibility of the isodimorphic crystal region to enzyme catalytic domain

39

165. Thus, it follows that biodegradation varies based on the crystallinity induced by the processing

method 166. Furthermore, based on the copolymer ratio, the amorphous region can be altered and

the enzymatic depolymerase activity can be maximized as seen in Figure 2.12.

Figure 2.12: a) PHB and PHBV crystallinity from WAXD patterns. b) rate of weight loss by 2µg of Ralstonia

pickettii type 1 PHA depolymerase due to 3HV ratio. Reprinted (adapted) with permission (Lic.

#4786580163872) from Feng et al. (2004) on March 12, 2020 167.

However, other studies indicate a PHA depolymerase is more effective on PHB than PHBV and

most effective on PH4B, although the results are measured in weight loss and the molecular

40

weights vary significantly 142. This indicates other enzymes or factors play a part in degrading

PHAs. For example, excess side chains may inhibit the rate of interaction with the target location,

and other actions coupled with the enzymatic hydrolysis can further enhance biodegradation.

Water diffusion, promoted by temperature, can induce some hydrolytic degradation which

increases active sites 168. Ester bonds of polyesters are also sensitive to hydrolysis and can result

in a reduction in molecular weight. The lowering of molecular weight invalidates the an Arrhenius

model for biodegradation 169, because enzymatic hydrolysis is coupled with ester hydrolysis and

overall biodegradation, and not driven by a single actuator. The depolymerisation steps are

indicated to be rate limiting steps in PHA biodegradation 170, thus enhancing any single form can

reduce biodegradable product life expectancy after its service life.

2.4.3.1 Crystallinity

The effect of PHA monomer units in the polymer structure is inextricably linked to the effect of

crystallinity that affects biodegradation. Polymer processing techniques also change the final

sample crystallinity, including solvent casting 171, and quenching 172, which either increase or

reduce the interlamellar phase and effect the long-term biodegradation extent. Although quenching

is not a concern due to PHAs low Tg.

As biodegradation proceeds, and mass loss increases, erosion of the interlamellar phase of PHA

begins in the initial stages 173, where the disordered chains are targeted first before samples are

eroded in the crystalline regions (Figure 2.13). The interlamellar phase or amorphous region gives

PHAs their flexibility, and with its degradation it is expected the crystallinity would increase.

These characteristics are seen within 30 days of soil biodegradation 174 and 60 days within marine

biodegradation 175. However, given enough time the crystallinity is expected to be reduced by up

to 60%, which occurs in soil biodegradation after 200 days or 30 days in a controlled composting

environment due to the intensified conditions 174. The amorphous region is known to allow

permeation of moisture and enzymes, and therefore its degradation would increase the surface area

of available crystalline regions. The crystalline regions make up the majority of a crystalline PHA

such as PHB and their degradation is expected to reduce the overall polymer crystallinity.

41

Figure 2.13: Schematic model of enzymatic degradation behaviour of lamellar crystal in P(HB) solution-grown

single crystal by PHB depolymerase. Reprinted (adapted) with permission from (Numata et al. H. Enzymatic

Degradation Processes of Poly[(R)-3-Hydroxybutyric Acid] and Poly[(R)-3-Hydroxybutyric Acid-Co-(R)-3-

Hydroxyvaleric Acid] Single Crystals Revealed by Atomic Force Microscopy: Effects of Molecular Weight and

Second-Monomer Composition on Erosion) on March 12, 2020. Copyright (2005) American Chemical Society

165.

2.4.3.2 Copolymers

Copolymers are heteropolymers where more than one type of monomer unit is present such as

PHBV, PH4B, PHBHx etc that contain HB units and another. Copolymers are known to

biodegrade faster, than homopolymers due to the presence of inherit amorphous region. For

example, PHBV is formed by addition of HV into PHB, where relative fractions of HB and HV

define modulating crystallinity. This lower crystallinity of the copolymer is directly related to a

42

higher enzymatic activity measured by PHBV weight loss. This corelation is reflected in the

degradation extent in both aerobic and anaerobic conditions. In the PHBV example, at 3% HV

content, there is no difference between PHB and PHBV degradation, as their crystallinity is

similar. As the HV content increases, the crystallinity decreases and the biodegradation rate

improves 176,177. Research indicates an HV content of approximately 40-50% produces the fastest

biodegradation rates in soil 170, compost 177, and marine water 146. The effect is also reflected in

anaerobic environments 15,178. Similarly, in other PHB copolymers, increasing the ratio of 4HB,

Hx, and HO relative to PHB correlates with a reduced crystallinity and melt temperature, compared

to neat PHB 179, and improves the biodegradation rate of the copolymers in aerobic (soil 180,

compost 177, marine 105,181) and anaerobic (sewage sludge 105,172, and anaerobic landfill 181)

conditions.

Another aspect to consider is the availability of enzymes to cleave the medium chain length PHAs.

Jendrossek identified several microorganisms having short chain length PHA degrading enzymes

182, and several short chain PHAs and other aliphatic polyesters such as poly(ethylene adipate)

(PEA), poly(ethylene succinate) (PES) and poly(2,2,6,6-tetramethylpiperidenyloxyl-4-yl

methacrylate) (PTMA) can be enzymatically depolymerized by similar enzymes, indicating a

significant advantage to short chain PHAs. These enzymes attach the ester bond but have low

specificity, not being overly discriminative toward the range of side chains 157. For example, PHB

has one carbon long side chain and some PHB depolymerases enzymatically hydrolyze PHBP 65,

which can be the result of PHBPs having no side chain. The enzyme affinity is likely determined

by the affinity towards the PHAs side chain 183. However, these enzymes do not function on

medium and long chain PHAs effectively. Research availability of medium chain length

depolymerases is minimal with very few species isolated with the particular enzymes 184, which is

further reflected by the few poly(3-hydroxyoctanoate-co-3-hydroxydecanoate) degraders seen in

Streptomyces 185. This may be why medium chain length polymers such as PHBO with 10% HO

content only show between 88-95% biodegradation in anaerobic and aerobic environments 181.

Thus, the use of high carbon PHA monomers such as 3-hydroxydecanoate in medium chain length

PHAs can impede biodegradability by reducing the number of chemically reactive binding sites in

the side chain structure for enzymes to attack.

43

The observed effect of crystallinity and copolymer ratio does not apply to all biodegradation

studies. Lower molecular weight polymers have enhanced motility and significantly reduced size,

which should make enzymatic degradation and bio-assimilation faster, but improvements are not

always reflected in solvent casting. The method of sample production (i.e. solvent casting) or

sample recovery method can similarly leave behind impurities or toxic residue depending on the

solvent 186 that may negatively impact the enzymatic activity or microorganism functionality and

produce irregular relationships between PHA copolymer compositions and their biodegradation

rates 170,187. Furthermore, there is evidence that PHB is a more readily used product than more

complex PHA monomers with larger side chains such as HV, 4Hb and Hx 147. Which is expected

to result in faster biodegradation rates, however, this is not always proven, and it has not been

determined if its sue to crystallinity alone.

2.4.3.3 Sample Morphology

The morphology of PHA samples is directly correlated to the surface area, where the

morphologically porous polymers have increased exposed surface area for enzymatic attack at any

given time which can increase the total enzyme binding sites. Therefore, a powder form has the

largest surface area to volume ratio and should have the fastest biodegradation. Some studies have

shown the PHB films can reach a comparable biodegradation rate, for certain film thickness to

PHB powder. Guttierrez et al. 188 reported PHB powder had 68% biodegradation in 12 days and

1.2mm PHB films had 67% biodegradation in 19 days. In another study, Sashiwa et al. 189 found

PHBHx/PBAT and PHBHx/PLA 440 µm powder and 20 µm films biodegradation had no

significant difference in anaerobic sewage sludge. It is not surprizing that thin films benefit from

enhanced biodegradation rates, due to the ease of moisture permeation and maximized surface area

190,191, such that their biodegradation rate improves as their thickness is reduced. It is encouraging

to see comparable results in both anaerobic 188 and aerobic 31 environments.

Biodegradation of films begins with surface pitting and degradation at the edges (Figure 2.14),

such that the surface area to volume ratio increases as time goes by. Furthermore, by changing the

production method of films from solvent casting to electrospinning, the surface area and water

permeability can be increased leading to a significant improvement in biodegradation rate,

44

measured by mass loss increase from 40% to 100% in a 28 day period 192. This method also

increases surface roughness, further enhancing surface area of thin films.

Figure 2.14: PHBV Film Surface at 35 days in Composting Conditions. Reprinted with permission (Lic.

#4786580681302) from Weng et al. (2010) on March 12, 2020 176.

Biodegradation of PHA pellets is generally slower than films to a certain extent depending on the

type of PHA. PHB films benefit more from thin film morphology compared to PHBV 150.

According to ISO 14855 PHB pellets biodegrade by 54% in 45 days and 92% in 78 days 193, which

is comparable to PHB plates of 1.2mm thickness 31. Therefore, when comparing biodegradations

studies, it is important to consider the morphology of the samples. However, the comparisons

become challenging as only a few completed studies followed ASTM standards.

2.4.4 Chemical Additives and Blending Effect on PHA Biodegradation

The addition of other chemical additives or blends all affect PHA biodegradation on both a

chemical and physical scale by impeding enzymatic degradation. Non-biodegradable co-polyesters

and chemicals minimize the interactable area between enzymes and biodegradable polymers. This

45

can be further exacerbated if the non-biodegradable polymer makes up the continuous phase with

PHA dispersed within 194, or the biodegradable polymer is unsuited for the particular environment

(i.e. PLA in marine water). This is mainly reflected in the rate of enzymatic hydrolysis.

2.4.4.1 Chain Extenders and Anti-fouling Agents

Chain extenders and anti-fouling agents are some of the most commonly applied chemical

modifications on polymers. As such, they are indicated to impact biodegradation at various

degrees, by inhibiting the extracellular enzymes activity or by inhibiting the microorganisms’

ability to produce the enzymes. For example, 4,5-dichloro-2-n-octyl-4- isothiazolin-3-one (DCOI)

is an anti-fouling agent, which is known to inhibit the onset of PHB and PHBV biodegradation

according to ASTM D5988 195. Chain extender Joncryl ADR-4368-CS, had a similar inhibitory

effect on the onset of PHA biodegradation, despite it being reported to reduce the crystallinity of

PHAs, which should improve the biodegradation 196 - at loadings of 5%, Joncryl chain extender

reduced the extent of soil biodegradation from 70 to 22%, in 340 days following ASTM D5988-

03 protocol. The inhibitory effect is also seen in composting conditions and marine conditions at

0.2 and 5% loadings, negatively impacting the biodegradation start time, rate and extent. Joncryl

is expected to have a scavenging effect which may sequester available electron acceptors, acting

as antifungal/antibacterial agent 149. Specific to chain extenders, the chain extension increases the

molecular weight and make PHAs more difficult to enzymatically hydrolyse. However, the extent

of chain extenders’ effects or different types have not been evaluated in other ratios with PHAs,

such as 0.5%, which is suitable for food packaging applications. PLA/PBAT blends with 0.5%

Joncryl-ADR-4368C have shown a 50% reduction in compost biodegradation extent in 126 days

197, and 0.5% Joncryl ADR-4370S and 1,6-hexanediol diglycidyl ether, similarly reduced the

hydrolytic degradation extent by 23 and 10% respectively 198. Therefore, there is strong evidence

chain extenders will reduce the biodegradation of PHAs.

2.4.4.2 PHA Blends

Biodegradation studies of PHA blends are not regularly completed in most natural environments

(i.e. soil and marine environments) due to the few other bio-based polymers showing evidence of

46

biodegradation such as PLA. Two main avenues of PHA blends have been explored, plasticized

PHA and PHA/PLA blends, due to their bio-based origins, as well as potential applications.

Plasticization of PHAs with tributyl citrate (TBC) or oxypropylated glycerol reduces the

crystallinity, but also slows the biodegradation rate in all blend ratios in soil, compost and

anaerobic sewage sludge 31,188,199. However, this may be due to the preservative effects (inhib its

microbial growth) of glycerols as a food additive, its reduced bio-assimilation due to

oxypropylation, and the synthetic production method and hydrophobicity of TBC 200,201. PLA has

been plasticized with acetyl tributyl citrate which inhibited hydrolysis action due to increased

hydrophobicity whereas triethyl citrate based plasticizers are more hydrophilic and can increas e

PLA degradation 202,203, and these effects likely play apart in reducing PHB degradation.

Furthermore, the way plasticizers are closely associated with polymer chains may inhibit enzyme

interaction with binding groups.

PHA/PLA blends similarly reduce the crystallinity and should result in improved biodegradation

of PHA components. However, PLA is not readily biodegradable in soil 204 and marine water 189,

due to the suboptimal temperatures 205. Some instances of 25-30% PLA in PHB, PHBV and PH4B

have shown evidence of similar or lesser, physical degradation or biodegradation rates in soil

conditions but no complete biodegradation is reported 174,206,207. However, according to ASTM

D5988-12, a PHA/PLA blend was seen to degrade to 99% in 176 days, but the ratio and presence

of additives were not identified 208. The addition of PLA, PBAT or PBS into PHBHx has also

resulted in a reduced biodegradation rate (3-23%, 10-90% and 20-90% respectively) compared to

PHBHx in marine water. The level of reported biodegradation is the result of PLA, PBAT and

PBS biodegradation in the blends, despite the virgin polymers not degrading in marine water 189.

There is no reported reason behind the unexpected biodegradation of PLA, PBAT and PBS in

marine water but it may be the presence of the highly amorphous hydroxyhexanoate 209, which

interferes with the crystallization of the other blend components or promoting biofilm

development.

Under higher temperature environments (~58 °C) such as controlled composting and anaerobic

digestion, the biodegradation of PLA is common, and it is expected there would be benefits of

47

incorporating PHAs into PLA to improve the compostability. However, PLA/PHBV 70/30 blends

have shown no biodegradation improvement compared to PLA and PHBV alone (all 90-92%

biodegradation in 200 days) under ASTM D5338-15 174, or have even reduced biodegradation rate,

under anaerobic digestion 210. The effects are similarly reflected in PLA/PHB/ATBC and

PLA/PHB/PEG based blends where disintegration properties were the same as virgin PLA or the

PHB is reported to slightly inhibit PLA degradation under ISO 20200 211. There is also evidence

that PHAs biodegrade slower than PLA in anaerobic digestion at 52 °C than PLA alone 210.

Considering the cost of PHA production being about double compared to PLA 79, it is not

economical to seek compostability or anaerobic digestion improvements with the addition of

PHAs. However, for mismanaged waste, industrial composting is not a suitable representation for

the natural environment and PLA is not a suitable for biodegradation in natural environments.

Overall, it has been established that PHA fully and rapidly (time frame) biodegrades in the natural

environment. Of the available studies few illustrate the biodegradability of PHAs in a comparable

manner.

2.4.5 PHA Soil Biodegradation

Recent PHA soil biodegradation studies are indicated in Table 2.1. Studies defined by mass loss

did not indicate (%) biodegradation based on CO2 evolution. Soil biodegradation is known to be

an incredibly dynamic environment that can vary across the globe. The process is considered

relatively slow compared to other aerobic and anaerobic processes, due to the mild conditions.

Cellulose for example can take nearly 200 days to degrade by 90% 193, however, if the conditions

are favourable, the reference material can take significantly less 212. Therefore, biodegradation of

PHAs will vary between studies.

PHAs are considered the most promising soil biodegradable materials, being 100% biodegradation

in 90 days, whereas PCL would take up to 270 days according to ASTM D5988 212. In non-ASTM

studies, PHBV is degrades by 40% in 120 days (25-30 °C) while no degradation was observed for

PBS and PLA 213. However, only 25-30% of microorganisms are PHA degraders, the rest only

bioassimilate the products of enzymatic hydrolysis 141. Furthermore, soil biodegradation rates have

48

been reported with several different conditions: (i) soil source; (ii) soil moisture content and pH;

and (iii) temperature of soil biodegradation studies. Based on literature, there is no standard that

defines a (%) biodegradation limit for polymers, to indicate whether it is biodegradable or only

shows biodegradable behaviour. The existing OK Soil degradable certification states it must be

90% biodegraded absolute or relative to the reference sample in 2 years under ASTM 5988 214, of

which few studies have continued for that long.

Table 2.1: PHA Soil Biodegradation Studies from 2010-2020.

PHA (co-

monomer %)

Protocol and Source Conditions Form Days Results Ref.

1 PHBV(12

% HV)

ASTM D5988-12,

Agriculture Field Soil

23-25 °C, 20%

Moisture (w.b.)

Film 200 35%

Biodegradation

174

2 PHBV(2% HV)

ASTM D5988-03, Forest Soil

25 °C Powder

350 70% Biodegradation

149

3 PHB Non-ASTM, Fertile

Garden Soil

30 °C, 80%

Relative Humidity, 10 cm Depth

Nano-

fibre Film

28 100% Mass

Loss

192

4 PHB Non-ASTM, Field

Soil

21 and 28 °C, 50%

Moisture (w.b.)

Film 35 60 and 95%,

Mass Loss

180

5 PHBV(12% HV)

90 and 100% Mass Loss

6 PHBHx(1

2% Hx)

92 and 100%

Mass Loss

7 PH4B(10% 4HB)

35 and 28

100 & 100% Mass Loss

8 PHB ASTM D5988, Natural Mature Soil

11-30 °C, 17-23% Moisture (w.b.)

Film 112 60% Mass Loss 195

9 PHBV(8% HV)

60% Mass Loss

10 PHB ASTM D5988-03, Commercial Soil

23 °C, 33% Moisture (w.b.)

Film 80 82% Mass Loss 59

11 PHA 76% Mass Loss

12 PHA ASTM D5988-03, Mixture of Topsoil,

Farm Soil and Sand

20 °C, 60% of Water Holding

Capacity

Film 660 70% Biodegradation

112

49

13 PHA Non-ASTM, Farmland Topsoil

35% Moisture (w.b.) Film 140 32% Mass Loss 190

14 PHA Non-ASTM,

Farmland Topsoil

35% Moisture (w.b.) Film 60 33% Mass Loss 215.


25 °C, 35% Moisture (w.b.)

Plate 120 35% Mass Loss 216


30-40% Moisture

(w.b.) Film 60 22% Mass Loss 191

2.4.5.1 Soil Source

According to ASTM standards, soil source need only be reported, and no specific location or soil

type is required. Its long since been known that soil type significantly impacts the degradation of

the most simple, short chain PHA polymers 217. However, no internal reference point has been

applied to give some form of comparability besides, the cellulose sample.

Measurements most often reference the location and the depth of the sample in the soil, and in rare

cases identify the microorganism community. However, the characteristics of the soil not only

affect the conditions but also how the conditions will change over the duration of the experiment.

For example, PHBV(8% HV) powder tested in sand (dry silica with low organic material) achieved

80% biodegradation after 600 days 175, which is significantly longer than most soil biodegradation

tests. Sand has relatively poor moisture holding capacity, substantiating these slow biodegradation

rates that take months to years for any evidence and by mixing in organic soil and farm soil into

sand, the biodegradation rate of PHAs are not seen to improve 112 (Table 2.1. Entry 12). The

degradation of PHBV(9.8% HV) film samples indicate minimal physical degradation after 5

months in a mixture of beach sand, horse manure and fertile soil 218. The effect of moisture holding

capacity is also reflected in Clarion loam soil 6% PHA biodegradation in 5 months after watering

once a week 219, even though clarion loam soil is rated to have very good lytic activity among

many other types of soil 124.

The limitations of sandy soil can be improved in natural environments near the coast, where

frequent water permeation happens continually. Such that the biodegradation of starch can be 4

50

times faster than garden soil 212. Other natural soil biodegradation studies include soul sourced

from South African agricultural fields, black soil and leaf mould, oil palm cultivation soil, soil

from Russia and Vietnam, and soil from California landfills. Field soil and landfill soil appear to

have the highest rate of PHA biodegradation activity, being the only samples approaching 100%

PHA degradation 141,143,150,174,180,204,220,221 (Table 2.1. Entry 1 and 4-7). This may due to the

microbial diversity found in moist soil with consistent environments that maximize microbia l

population growth and diversity.

The location of soil in a specific area is of significant impact on the potential biodegradation rate.

Between subsoil and topsoil, fungi community is richer in topsoil 222 and therefore the

biodegradation rate with increased microorganisms will be significantly higher. Variations in the

natural environments such as humidity under larch and birch trees, and in harsh winters over a

yearly basis were reflected in lower microorganism counts and slower mass loss due to lower

humidity 143. The environment fosters the microorganisms, comparing PHA degradation in

greenhouse soil and farmland soil it was found PHA degradation is significantly greater in farm

land soil for similar sized films/plates under similar conditions 191,215,216 (Table 2.1. Entry 14-16).

Celestina et al. 222 reported greenhouse soil treated with fertilizer has a lower bacterial and funga l

community count and diversity compared to untreated soil. Therefore, it is important to note the

soil source and conditions being used during the biodegradation study of PHAs.

2.4.5.2 Soil Moisture Content and pH

The enzymatic hydrolysis of PHAs is coupled with the moisture content to properly hydrolyse the

ester bonds, and therefore high moisture content maximizes PHA enzymatic and biodegradation

performance. For example, PHBV can degrade in soil by 30-40% in 120 days provided there is

enough moisture 213, but in arid “dry” environments, PHBV degradation can extend as biotic and

abiotic processes slow. In the literature, it’s referred to as either water holding capacity (%) or soil

moisture/humidity (%). According to ASTM 5988-96, at 90% water holding capacity, PHB

powder can degrade by 100% in 90 days 212. Sand has incredibly poor moisture holding capacity

as it is almost entirely based on silica oxide. Soil biodegradation studies without a measure of

moisture content are considerably older since the standard has been updated to include soil

51

moisture. Comparing similar soil studies using field soil at 28 °C and 50% moisture content to

fertile garden soil at 30 °C and 80% moisture content, PHB films take 28 and 35+ days to achieve

100% mass loss in optimal microbial conditions, respectively 180,192 (Table 2.1. Entry 3 and 4).

PHBV(12% HV) only biodegrades by 35% in 200 days at 23-25 °C, which can also be attributed

to the 20% moisture content 174 (Table 2.1. Entry 1).

Soil pH is closely tied to moisture and the degradation of PHAs, by maximizing enzyme function.

Optimal pH for the most common enzymes used in PHB degradation is between 6-9 with some

variation between different types 223. Furthermore, PHB and PHBV ester hydrolysis and physical

degradation naturally occurs in alkali environments without microorganism presence 100. Few

studies consider these points, however, it has been indicated the optimal soil for PHB degradation

is saline soil with a pH of 7-8 compared to clay, sandy, tarine, and laterite soil with a pH lower

than 7 224. The effect of soil pH on PHAs is similarly caused by alkali treating the PHB to improve

the hydrophilicity and initiation time of mass loss at the cost of slightly reduced mechanica l

properties 225. PHA degradation is maximized with high moisture and optimal pH range, however,

this does not capture the soil pH and moisture relationship around the world. 50% of the tested

regions in North and South America have an acidic surface soil pH (<6.5) and evidence of a

positive correlation between moisture content and acidic pH indicate ideal conditions for natural

PHA degradation is very limited 226.

2.4.5.3 Temperature Effect in Soil

Enzyme kinetics is directly correlated to temperature, with best rates observed at optimal

temperatures for the microorganisms and the set of enzymes. Several studies have since shown

that soil biodegradation at higher temperature ranges proved more favourable. In hardwood soil,

at temperatures of 15, 28 and 40 °C, PHB degradation increased to 8%, 23% and 26% respectively

178. In other studies PHB degradation at 30 °C had a 100% improvement over degradation at 20

°C 224.

For standard purposes, soil biodegradation should be completed at 20-28 °C, but do perform better

at higher temperatures. PHA copolymers have greater thermal sensitivity during biodegradation;

52

PHBV(10% HV) degradation at 15, 28 and 40 °C were 11%, 32% and 100% respectively over

approximately 200 days 178. The degradation rates of PH4B(10% 4HB), PHBHx(12% Hx),

PHBV(12% HV) and PHB have all seen degradation improvements as the study temperature is

increased from 21 to 28 °C, with all copolymers benefitting the greatest with increased

temperatures 180 (Table 2.1. Entry 4-7). PHA molecular mass reduces faster at higher temperatures

in soil, suggesting it would benefit more from composting conditions. In most cases <40 °C gives

the best improvement in PHA degradation rate (due to microbial action or enzyme activity) while

temperatures approaching 60 °C were significantly worse for PHA degradation 227. This can be

related to type of microbial species and proteins present and their respective functions as there is

a shift from mesophilic to thermophilic microorganisms at 40 °C 127, in addition to PHB

depolymerase activity reducing at temperatures increase from 30 to 70 °C 223. In soil

biodegradation low temperature (<20 °C) studies are not regularly completed, limiting the

available literature on biodegradation in cooler climates and environments.

2.4.5.4 Applications for Soil Biodegradable PHA

The desirable soil biodegradation properties of PHAs make them an excellent candidate for single

use plastic applications that are common in plastic waste, including for biodegradable plastic

applications in agricultural industry. For example, PHA based mulch films can be supplemented

with nutrients and other additives that can be slowly released, timed by the degradation of the

mulch films. Furthermore, some mulch films can assist in moisture retention by protecting the soil

from moisture evaporation. Redondo et al. 208 studied a PHA/PLA blend in commercial soil under

ASTM D5988-12 where mulch films degraded completely after 176 days, covering a considerable

amount of the growth season. However, the environment of surface soil is not generally hospitable

to biodegradation action. UV degradation does not assist PHA mulch film degradation and the low

moisture with high temperatures make it unfavourable for microbial degradation 228.

Considering there is no standard that defined whether a polymer can be claimed soil biodegradable

after achieving a certain extent of degradation, it is difficult to define whether PHAs are soil

biodegradation. However, there is enough evidence to claim in literature that PHAs are soil

53

biodegradable provided the conditions and sample morphology are appropriate, but the duration

may vary anywhere from 20 days to several hundred or more.

2.4.6 PHA Composting

Industrial Composting is characterized by the controlled temperature of 58 °C, and can be

completed industrially. Most studies are completed under industrial compost conditions, as home

composting doesn’t consistently achieve this high temperature level. Under ISO 14855 (equiva lent

to ASTM D5338) composting conditions, cellulose degrades by 92% in 45 days 193 (Table 2.2.

Entry 15). Comparatively under ASTM 5338, a PHA bag has 94% biodegradation in 180 days 38

(Table 2.2. Entry 1), and exceeds the degradation of petroleum based biodegradable polymers like

PCL 229, and passed the 2nd tier of the ASTM 6400-19 compost labelling standard which is 90%

biodegraded in 180 days. PHA composting studies within the last decade are outlined in Table 2.2.

Although industrial composting is considered very controlled, there is still some variability

between samples, PHBV films has been reported to take between 45-200 days (Table 2.2. Entry 2

and 3). This variability may have to do with the inoculum source.

Table 2.2: PHA Composting Biodegradation Studies from 2010-2020.

PHA (co-monomer %)

Protocol and Source Form Days Results Ref.

1 PHA ASTM D5338 Bag 180 94% Biodegradation 38

2 PHBV(12% HV)

ASTM D5338-15, Mushroom Compost

Film 200 90% Biodegradation 174

3 PHBV(2% HV) ASTM D5338 Film 45 95% Biodegradation 149

4 PHB ASTM D5338-98 0.24 mm Plate

112-140

99-100% Mass Loss 31

5 1.2 mm Plate 84-112

98-100% Mass Loss

6 5 mm Plate 210 45% Mass Loss

7 PHB ISO 14855-1, Compost Factory Organic Waste


8 PHBV(3% HV) 80% Biodegradation

9 PHBV(20%

HV)

89% Biodegradation

10 PHBV(40% HV)

90% Biodegradation

54

11 PH4B ISO 14855-1, Compost Factory Organic Waste


12 PHB ISO 14855-1, Mature

Compost



14 PHB Non-ASTM, Home

Composting

Tensile

Sample

84 50% Mass Loss 230

15 PHB ISO 14855, Mature Organic Municipal Solid

Waste

Pellets 78 92% Mass Loss 193

16 PHB ASTM D5929-96 0.5 mm Plate 182 100% Mass Loss 31

17 1.2 mm Plate 182 100% Mass Loss

18 3.5 mm Plate 350 94% Mass Loss

2.4.6.1 Inoculum Source in Composting

The inoculum source for composting is the material used to introduce the microorganism into the

compost that will be degrading the materials. ASTM D5338 indicates the inoculum can be from

municipal solid waste, plant waste, yard waste or a mixture of green waste and municipal solid

waste. In such cases some variability can be introduced. PHA biodegradation has been studied

using inoculum sources from activated sludge 231, mushroom farm compost 174 (Table 2.2. Entry

2), organic waste from composting factory 177 (Table 2.2. Entry 7-11) or mixtures of chicken

manure with wood chip dust 232. Having no defined ASTM standards, the compost init ia l

conditions (pH, carbon/nitrogen ratio etc.) have significantly more variability between studies.

Comparing activated sludge, and chicken manure mixed with wood chip dust as an inoculum

source, activated sludge inoculated compost had significantly higher biodegradation rate 231,232.

Activated sludge is sourced from wastewater treatment and inherently contains a variety of bacteria

and protozoa that can vary across the globe in terms represented microbial populations and more

locally, depending on the treatment methods.

Following ASTM D5338-15 or its equivalent ISO 14855-1, a comparison of organic waste

inoculum and mushroom compost inoculum indicate organic waste is more effective 174,177 (Table

2.2. Entry 2 and 11). Organic compost is characterized by a high moisture content from the organic

55

material improving the conditions for microbial growth and its starting microbial load is

significantly higher than inoculum sourced from the mushroom compost.

2.4.6.2 Non-Commercial Compost (Home)

Home or non-commercial composting is a more natural method where the temperature and

moisture content is not controlled, allowing for more variation in the initial compost composition

and temperature profile. As such, home composting performs slower than commercial or industr ia l

composting.

Biodegradation of PHAs under home composting or industrial composting follows the pattern of

other biodegradable plastics studied under the same conditions, influence by the microbial profile,

temperature and pH during the process. PHAs show minimal if any biodegradation in home

composting conditions where the temperature is low, or when the pH becomes low 229. Higher

temperatures of industrial composting benefit biodegradation by enhancing non-enzymatic and

enzymatic catalysed hydrolysis 233. Because of these variable conditions, PHA biodegradation

varies between each study. Mergaert et al. 178 reported biodegradation of PHB, PHBV(10% HV)

and PHBV(20% HV) to be 4%, 6-17% and 67% in 152 days where the temperature varied from

8-30 °C. Gilmore et al. 144 reports PHBV(26% HV) had 59% mass loss in 186 days, indicating a

slower biodegradation which is slightly slower despite the temperature between 40-63 °C

throughout the study. The difference is more likely due to differences in inoculum and temperature

profile which is reflected in PHB which degrades by 50% in 84 days in organic waste home

compost with 74-89% humidity and temperature of 34-66 °C 230 (Table 2.2. Entry 14). The

unpredictability of PHA home composting makes it difficult to study the effects of temperature

profile throughout the composting process.

Several composting studies (Table 2.2. Entry 1, 3-5, 7-11, 15-17) following ASTM D5338 or ISO

14855 have identified PHB and PHBV to pass the 1st and 2nd tier of ASTM D6400. Samples are

90% physically degraded in 90 days, defined by (%) mass loss, and 90% biodegraded in 180 days,

defined by (%) biodegradation.

56

2.4.7 PHA Marine Biodegradation

PHAs are the only class of bio-based polymers that exhibit efficient marine biodegradation,

compared to other polymers such as PLA – which does not degrade 234. Among other polymers

showing biodegradable behaviour in marine water (i.e. PCL, PES, PEA etc.), PHAs outperform

most in all water environments 187, but the research in PHA marine biodegradation following

repeatable standards is still very limited. Recent PHA marine biodegradation studies are outlined

in Table 2.3. Over 70% of the world is covered in water and presents a variety of differ ing

conditions in the natural environments which must be investigated to ensure PHA waste is

biodegraded if improperly disposed.

Table 2.3: Recent PHA Marine Biodegradation Studies.

PHA (co-monomer %)

Protocol and Source Conditions Form Days Result Ref.

1 PHB Non-ASTM, Eutrophic Reservoir

18-25 °C Film 42 43.5% Mass Loss 99

2 PHB Non-ASTM, South China Sea

27-30 °C Solid 160 62% Mass Loss 145

3 Film 58% Mass Loss

4 PHBV(11%

HV)

Non-ASTM, South

China Sea

27-30 °C Solid 160 87% Mass Loss 145

5 Film 54% Mass Loss

6 PHB ASTM D6691,

Woods Hole Harbor Water

30 °C Film 100 90% Mass Loss 163

7 PH4B(44%

4HB)

ASTM D6691,


30 °C Film 100 80% Mineralization 163

8 PH4B(47% 4HB)

82% Mineralization

9 PHBV(8%

HV)

ASTM D6691,


30 °C Film 100 85% Mineralization 163

10 PHBV(12% HV)

100% Mineralization

11 PHBV(8%

HV)

Non-ASTM, Lorient

Harbour


12 PHBV(8% HV)

Non-ASTM, Lorient Harbour Water +

Foreshore Sand

25 °C Powder

210 90% Biodegradation

175

13 PHA 2200 ASTM D6691-09 30 °C Film 365 52% Biodegradation

234

57

14 PHA 4100 82% Biodegradation

15 PHBV(12%

HV)

Non-ASTM, Baltic

Sea Water

17-20 °C Film 42 60% Mass Loss 231

16 PHBHx(6.5% HV)

Non-ASTM, Coastal Seawater

23 °C Film 148 89% Biodegradation

105

17 PHBHx(7.1%

HV)

195 55% (*77%)

Biodegradation

18 PHA Non-ASTM, Tropical River Water


19 PHBHx(11%

HV)

Non-ASTM, Sea

Water

27 °C Film 28 35%

Biodegradation

189

20 PHBV OECD 301, river water

25 °C Film 90 90% Biodegradation

149

*biodegradation level after removal of outlier in data for sample

2.4.7.1 Marine Water Source

Marine water source can come from various sources (i.e. oceans, lakes, rivers etc.) but saltwater

from ocean sources is usually classified as marine water and will be considered as such in this

study. However, some studies have been completed in fresh water, static water, river water or a

combination thereof. Marine water has a higher level of sulphates (compared to fresh water) which

can act as a secondary terminal electron acceptor for some microorganisms. The pH of fresh water

is usually lower than marine water 181, which does not benefit PHA biodegradation. Furthermore,

the majority of aquatic microorganisms are bacteria while fungi are found considerably less often

in large water bodies across the globe 104. Knowing fungi are considered more efficient PHA

degraders, the marine biodegradation performance of microorganisms will be slightly slower.

In certain studies, a combination of marine water and sand have been used to study the effect of

the different microorganism’s biodegradation performance. PHBV shows biodegradation in both

water and sand alone as well as a combination. The presence of marine water is seen to promote

biodegradation, which suggests a wide distribution of PHA degraders in water or more effective

enzymatic hydrolysis. PHBV is reported to take 210 days to degrade by 90% in a sand and seawater

medium, but over 600 days to degrade by 80% in only sand 175 (Table 2.3. Entry 11 and 12). During

58

Non-ASTM studies in non-laboratory conditions, Sridewi et al. 132 reported degradation of PHAs

is high where tide inundation is higher due to the presence of water and the introduced microbia l

populations not found in the soil alone. However, The improvement may be attributed to the

presence of water and not the microorganisms in it, because sand based environments are reported

to have a greater Shannon-weaver diversity index and overall diversity of phylum compared to

water environments 236. Therefore, sediment provides a great microbial diversity while marine

water may significantly enhance the enzymatic function.

Other inoculum sources for non-natural marine biodegradation include sewage sludge and

anaerobic digester sludge. Anaerobic digester sludge outperforms sewage sludge as an inoculum

source for marine aerobic biodegradation of PHAs 237.

2.4.7.2 Temperature Effect on Marine Biodegradation

The effect of temperature in marine biodegradation reflects temperature-dependent findings from

soil biodegradation studies 146. In cold water (at approximately 5 °C) the biodegradation of PHAs

is negligible 238, indicating that cooler water bodies located in the far northern or southern

hemispheres are less conducive to supporting biodegradation. At milder temperatures (between

10.9-19.8 °C), PHA biodegradation in water was slower at lower temperatures, during the init ia l

phase of the study 175. The initial phases of biodegradation affect the microbial growth

characteristics, which may require biofilm development before more rapid biodegradation

proceeds. The optimized degradation temperature for PHB in fresh water (no data for other PHA),

is indicated to be 30 °C, doubling the degradation rate when compared to 20 °C. Water temperature

of 40 °C did not further improve PHB degradation rates 224. Based on this data, PHA plastics would

be a clear benefit to curb the plastic pollution in tropical environments, including the garbage

patches floating in the North Pacific and Atlantic Ocean 21–23.

2.4.7.3 Natural Marine Environments

Natural marine environments are mainly characterized by their dynamic nature, integrating a

continuous ecosystem, opposed to a batch or static process in the laboratory. There are strong

indications that dynamic sea water assists in degradation of biodegradable polymers relative to

59

static sea water in the lab environment 239. Closed experimental systems also limit the potential

biodegradation rate by having accumulation of by-products as well as limited nutrients available,

which may result in lowering the pH and slowing the PHA break down into acids etc. A study of

a PHA sample in tropical marine water studied under static conditions, observed the reduction in

the pH of the system from 7.5 to 4.7 and degradation only up to 71%, after which degradation

stopped due to the acidic pH 235 (Table 2.3. Entry 18). The acidification limited the

enzymatic/hydrolytic degradation of the long chain PHA polymers. Furthermore, static

environments promote the development of biofilms more readily compared to dynamic

environments. Deroiné et al. 175 reported the effect of 0, 5% and 50% biofilm (fish breeding tank

wall biofilm concentration in marine water) on the biodegradation of PHBV(8% HV) and found

no biofilm had 36% mass loss in 180 days (Table 2.3. Entry 11), 5% biofilm had 97% mass loss

in 200 days and 50% biofilm had 88% mass loss in 300 days. Excessive biofilm can produce an

overabundance of enzymes which can cover the active sites for enzyme binding to perform

hydrolytic degradation. Current marine biodegradation standards do not capture the full

advantages of observed natural marine biodegradation rate.

Natural marine environments can also include fresh water sourced from rivers and lakes, where

biodegradation of plastics may occur. Compared to other biodegradable polymers, PHBV

biodegrades the most efficiently in many aquatic environments. PHB and PHBV biodegrade at a

different rate in aquatic environment, having 6 times faster rate in seawater than fresh water

ponds/canals 178. Under favourable conditions, PHA can be degraded in its entirety, within 28 days

in both fresh and saltwater rivers/lakes/oceans. PHB and PH4B degrade slightly slower within the

28-day period. Overall, chemically synthesized polymers tend to degrade at a slower rate and to a

lower extent and are more dependent on the origin of water: PCL and PEA have comparable

biodegradable behaviour in fresh and salt water to PHAs; PES biodegrades well in fresh water

only; and PBS and poly(butylene adipate) (PBA) show some biodegradable behaviour in fresh

water and minimal in salt water 187. In terms of the scope of ocean saltwater, PHAs are well placed

to replace plastics that may end up in the ocean. However, at a considerably greater ocean depths,

PHAs degrades slower 140.

60

While marine biodegradation does not contain an equivalent labelling standard to ASTM D6400,

under ASTM D6691 PHB, PHBV(8-12% HV) and PH4B(47% 4HB) biodegrade beyond 90% in

100 days relative to the glucose reference 163 (Table 2.3. Entry 6, 8-10), fulfilling part of the OK

marine biodegradable standards 136. Therefore, PHAs are marine biodegradable under ASTM

D6691 in appropriate environments but physical degradation must still be assessed.

2.4.8 PHA Sewage Sludge Biodegradation

Sewage sludge biodegradation is the industrial biodegradation of sewage sludge in munic ipa l

waste facilities, and sewage sludge biodegradation studies use sewage sludge as an inoculum.

However, there is little rationale why PHAs would be found in sewage sludge. Biodegradation in

sewage sludge is usually completed at mesophilic temperatures of 35 °C in a non-industrial scale

to stabilize sewage sludge for land application, allowing many types of biodegradable polymers to

degrade rapidly. Sewage sludge biodegradation is classified by low total solids required in the

inoculum, and is more representative of anaerobic water biodegradation. In these conditions PHA

are still reported to degrade into 50% CO2 and 50% methane 240, and at least 2 times faster than

other biodegradable polymers (PBS, PLA and PCL) 181,241. Standards of this kind have been

withdrawn as anaerobic digestion with higher solids is a more applicable and representative form

of anaerobic biodegradation used in sewage sludge processing on an industrial scale. PHA

anaerobic sewage sludge biodegradation studies are outlined in Table 2.4.

Sewage sludge biodegradation varies significantly but approximately 40 days is required for PHB

films to biodegrade to 90% under ASTM D5210 (Table 2.4. Entry 5 and 9). Following Non-ASTM

standards in laboratory conditions, PHA biodegradation can occur in as little as 16 days.

Table 2.4: PHA Anaerobic Sewage Sludge Biodegradation Studies.

PHA (co-

monomer %)


1 PHB ISO 13975, Anaerobic Sludge

Powder 10 90% Biodegradation 148

2 PHBHx(6.5%

Hx)

Non-ASTM, Wastewater Film 85 55% Biodegradation 105

3 PHBHx(7.1% Hx)

77% Biodegradation

61

4 PHB ASTM D5210-92, Diluted Sewage Sludge


5 Plate 19 68% Biodegradation

6 3 PHBs (Commercial or

Research)

Non-ASTM, Synthetic Sludge with Anaerobic

Biomass

Powder 40 50-79% Biodegradation

242

7 PHBO(10% HO) Non-ASTM, Wastewater and Septic Sludge


8 141 95% Biodegradation

9 PHBV(8% HV) ASTM D5210-91,

Wastewater


10 PHB Non-ASTM, Domestic Sewage Sludge


11 PHB Non-ASTM, Sewage

Sludge




2.4.8.1 Inoculum Source of Anaerobic Sewage Sludge

The inoculum of anaerobic sewage sludge largely defines the microorganism community that

develops in the digestor and ultimately determines the biodegradation pathways developed and the

final products. Under anaerobic conditions, sewage sludge microorganism communities are able

to produce a greater amount of CO2 relative to methane, compared to fresh water and marine water

181. Therefore, inoculum from anaerobic sewage sludge digesters have been applied for wastewater

treatment plants and biomass biodigesters. Of the potential sources, inoculum from mesophilic

(<40 °C) microorganism consortia are more effective in breaking down and converting PHAs,

being best suited to the environment in a sewage sludge anaerobic biodegradation. Conditions from

waste water treatment plants can be thermophilic (55 °C) and the thermophilic microorganisms

and their enzymes may not be suited to function in mesophilic conditions 240–242. Comparing

inoculum sources of fresh water sediment and marine water, there is no significant difference

between the biodegradation extent 181.

62

2.4.8.2 Products of Anaerobic Sewage Sludge

The products of anaerobic sewage sludge have been evaluated for biomethane production to

maximize the methane production for industry. The anaerobic biodegradation PHB produces

biogas with 80% methane, reaching a theoretical conversion yield of 60% of the total carbon

available in the polymer 137 (Table 2.4. Entry 10). PHAs with higher HV content, lead to more

rapid methane yield, however, there is no significant difference in final % methane produced

between PHBVs of different HV contents 15. Shin et al. 240 reported PHBV(8% HV) has 50% of

the biogas produced is methane (Table 2.4. Entry 9). Production of methane can occur depending

on the inoculum and the substrates. It has been found marine water produces higher methane

content as biogas compared to fresh water sediment as an inoculum 181 (Table 2.4. Entry 7 and 8).

Methane production is increased in co-digestion of PHAs with waste organic matter 242 (Table 2.4.

Entry 6). Modifying the methane production during anaerobic biodegradation has the potential to

improve the value of anaerobic biodegradation compared to aerobic biodegradation because

methane can be captured and utilized instead of being released in the environment as a greenhouse

gas. However, not all anaerobic processes are controlled and managed by industry. Therefore,

anaerobic sewage sludge conditions may vary significantly, and are expected to impact the PHA

biodegradation

2.4.9 PHA Anaerobic Digestion Biodegradation

Anaerobic digestion is a part of the wastewater treatment utilized to reduce the organic matter

content present in its effluent. The process can be either thermophilic (55°C) or mesophilic (35

°C) and can be used to manage waste or produce biomethane. For the disposal of polymers, it is a

suitable method to produce methane from food contaminated materials that can’t be recycled.

Under ASTM D5511 PHA biodegrades entirely in 20 days under mesophilic conditions 38. PLA

and PCL both require a temperatures above 37 °C to show any form of biodegradation in anaerobic

conditions 148,241. While thermophilic anaerobic digestion is faster and more beneficial in methane

production, mesophilic anaerobic digestion is more common in industry due to energy input 243.

Under anaerobic conditions, PHAs degrades faster at mesophilic conditions, while at thermophil ic

temperatures, PLA degradation is faster 210. Exploring mesophilic anaerobic biodegradation for

63

PHAs has significant potential as a waste disposal method if plastic ends up in the wastewater

treatment systems.

2.4.9.1 Product Ratio Modification in Anaerobic Digestion

The products of anaerobic digestion are significantly important for industrial bio-methane

production through the biodegradation of organic matter. PHA biodegradation normally yields 50-

60% methane from the total theoretical carbon yield, or approximately 80% methane from the total

biogas yield 241. This is comparatively lower than PLA and PBS yields. Co-digestion has also been

studied in conditions like anaerobic digestion, where food waste and PHA film were co-added.

The methane production can be maximized in 45 days, however, during this period, no or limited

PHA biodegradation occurs due to glucose repression244. Despite the increased methane yield, the

lag in onset of biodegradation defeats the point of using PHAs for that feature.

2.4.9.2 Temperature Effect in Anaerobic Digestion

The effect of temperature on anaerobic digestion is correlated to the enzyme activity. It is well

established that mesophilic enzymes function best at 30 °C, and their activity linearly reduces until

70 °C, were no activity is reported 223. According to ISO 13975, PHB powder degrades by 90%

in 10 days at 37 °C, while PLA, PBS and PCL had <43% in biodegradation in 277 days. At 55°C

PHB had 83-98% biodegradation in 22 days while PCL and PLA had 82-84% biodegradation in

96 days and PBS did not degrade. There is evidence that PHB degrades slower in thermophil ic

conditions 148,241, however, this characteristic is not evident in all studies. Hegde et al. 210 reported

PHA/PLA blends degraded 40% slower than virgin PLA at 52 °C in 60 days, and PHA only helped

initiate the degradation of PLA. Furthermore, there is evidence that PHB degrades faster in 30 °C

over 40 °C anaerobic sewage sludge 224, and even faster under aerobic digester conditions 237.

Therefore, it is reasonable to conclude PHAs are suited for applications where the disposal is in

natural temperature range conditions where PLA, PCL and PBS show minimal, if any, degradation.

64

2.4.10 PHA Accelerated Landfill Biodegradation

Accelerated anaerobic landfill conditions are under mesophilic temperatures (35 °C) with a high

ratio of solids, reflecting an environment much like a landfill. Landfill conditions are usually

uncontrolled due to the quantity of material flowing into landfills continuously. Under optimal

conditions and a high enough moisture content, PHB will degrade completely within 9 days, while

the more complex PHBV may take longer to bio-assimilate (29% biodegradation in 42 days) after

being degraded 147. The bio-assimilation is mainly based on the microorganism consortium.

2.4.10.1 Inoculum Source of Anaerobic Landfill

The inoculum sources greatly affect the biodegradation rate under landfill conditions. Weaver 245

characterized the effects of different inoculum sources for anaerobic landfill digestion conditions

and found that among a landfill reactor, waste water treatment plant, landfill leachate and

anaerobically digested organic waste. The digestate and municipal solid waste provided the most

reproducible results and also found the inoculum sources significantly affect the rate and extent of

biodegradation. Both the digestate and waste from a municipal solid waste plant, have the

advantage of being from a continuous system where the microorganism turnover is continuous ly

occurring, and the organic material promotes growth conditions.

Moisture content is another factor. Under a high enough moisture content (50-90%), PHAs have

been reported to completely degrade within 14 days 147,172. However, when the moisture content

is below 50%, biodegradation extent is halved and biodegradation can exceed 180 days 181.

The main limitation of PHA anaerobic landfill biodegradation analysis is the few available studies

being completed following either ASTM D5526-18 or ASTM D7475-20. Furthermore, while there

is consistent evidence that PHAs show favourable anaerobic biodegradation behaviour, there is no

labelling standard to define whether it can be claimed as such and it is not desirable to landfil l

biodegradable plastics unless the off-gases can be captured.

65

2.4.11 Conclusions of Biodegradation

Several factors affect the biodegradation of PHAs in the natural environment (non-laboratory)

including location, temperature, nutrients, microorganisms present, UV light exposure, dissolved

oxygen and salinity. In lab scale studies, optimal temperature, moisture content, pH and higher

amorphous content consistently promote enzymatic depolymerization of PHAs in all types of

aerobic and anaerobic biodegradation. The bio-assimilation of PHAs is mainly depends on the

complexity of the polymer, where less complex polymers are more easily assimilated, but at the

same time are limited by their high crystallinity (that limits degradation rate). Furthermore, the

presence of other organic matter can either promote the bio-assimilation of PHA to produce

specific products (methane) or can repress the bio-assimilation of PHA in favour of more easily

accessible carbon sources (e.g. cellulose, starch, glucose etc.). Mesophilic temperatures are

reported to be most favourable for PHAs compared to other biodegradable polymers (i.e. PLA), in

both aerobic and anaerobic biodegradation.

The effect of additives and blends of PHAs are important to consider, since virgin PHAs are not

usually used in industry. Chain extenders, antifouling agents and the synthetically produced

plasticizers TBC and glycerol, inhibit the biodegradation of PHAs, despite reducing the

crystallinity, either by inhibiting the enzymatic action, reducing hydrophilicity or inhibiting the

microbial growth. In such cases these can delay the onset of biodegradation for tuneable attributes

if so desired.

Inoculum sources provide a significant added diversity of microorganism consortia that is

responsible for variability of PHA biodegradation between studies. In high solid content studies,

such as soil biodegradation, marine biodegradation and landfill biodegradation, a high moisture

content allows for the microorganisms to have an increased diversity and population that can

enhance the biodegradation rate. Therefore, to ensure biodegradation studies are applicable in

research, they should follow ASTM/ISO standards, and also have inoculum sources that are

comparable to either a natural environment or comparable inoculum sources that are readily

available such as those from waste water treatment plants.

66

2.5 PHA-Based Biocomposites

Biocomposites applications exist mainly in the automotive, packaging, and consumer product

industries to reduce material costs and weight. Biocomposites can have improved impact and

mechanical modulus compared to the virgin polymer. However, the effects are dependent on the

fibre modulus, aspect ratio, morphology and interfacial adhesion of the fibre to the polymer, in

addition to the properties of the polymer itself that are being reinforced. The impact and/or

modulus do not always improved if the properties of the fibre are lower than the virgin polymer

246. The use of biocomposites introduces a sustainable application for agricultural fibres when their

service lives end aside from disposal 10. Cotton for example is still the most produced fibre today

247, and is the most basic natural fibre consisting of predominantly cellulose. Other types of fibres

contain cellulosic material such as hemicellulose of lignin depending on the source. In fibres,

lignin acts as the cement and cellulose is the rigid structure that gives the biocomposite the

increased moduli 248. PHA biocomposites are composites using natural fillers instead of inorganic

fillers, it’s important to consider the composition of the natural fillers and fibres for biodegradation

purposes because they need to be degraded during the process.

2.5.1 Natural Fibres and fillers

Natural fibres and fillers, composed of organic material are essentially composed of cellulose,

hemicellulose and lignin, with some other comonents depending on the type such as protein, starch,

silica and other impurities. Natural fibres are usually composed of the first three, which results in

a structure seen in Figure 2.6. Variation of these fibre ratios results in different types of fibres

(bast, leaf, grass, straw and seeds). Several PHA-based biocomposites have been developed using

kenaf 220, abaca 249, flax 250, starch 140, wheat straw 173, sisal 251, hemp 252, cellulose 253, lignin 149,

seagrass 254, wood flour 164, etc. 191,254–257. However, in many cases it is always assumed that the

addition of fibre improves the biodegradation of PHAs.

67

Figure 2.15: Basic Natural Fibre Structure.

2.5.2 Compatibilizers/Coupling Agents

Despite the hydrophilic favourable properties of natural fibres, making them relatively more

compatible with polar biopolymers, compatibilizers are usually required to enhance the shear stress

between fibre and matrix.

Maleic anhydride (MA) is one of the most common fibre-biopolymer grafting methods, although

others such as silane treated fibres have been successfully used as well 258. MA grafting does hold

other benefits such as reducing odour release during processing 259. However, MA can target the

oxygen species in polymers, thus enabling it to functionalize the carboxyl and hydroxyl groups in

biopolymers and the hydroxyl groups in cellulose, lignocellulose and hemicellulose 260. This is of

particular concern for biodegradation because of the hydrophilicity associated with polar carboxyl

and hydroxyl groups.

In PHBV, MA grafting occurs predominantly on the PHB molecule, due to the relative number

and chemical/statistical/steric effects. The ethyl group on PHV hinders reaction and reduces

acidity, decreasing availability of hydrogen atoms. Figure 2.16 illustrates the reaction scheme

between PHBV and MA 261, where the enzyme availability of HB may be inhibited.

68

Figure 2.16: Reaction scheme between PHBV and MA in presence of an initiator. Redrawn with permission

(Lic. #4786581071720) from Avella et al. (2007) on March 12, 2020 261.

2.5.2.1 Grafting Effect in Biodegradation

However, some chemical additives or polymers interfere with enzymatic actions upon the fibre.

Of greatest concern is grafting compounds into PHAs, which fundamentally change the chemical

structure and may limit the enzyme function. Several studies of the biodegradation of PHA films

grafted to 10 wt.(%) acrylic anhydride (AA) or MA have been completed in field soils and

greenhouse soils. An increase in the mass loss (%) by 3-5% was observed with the addition of

maleic anhydride or acrylic anhydride. The indicated location of grafting for both anhydrides is on

the butyrate side chain of PHB, which is expected to reduce the availability of the primary PHA

degradation target and inhibit enzyme action. However, the crystallinity of all PHAs are slightly

reduced with the grafting and the water absorption increased, as a result of either the increased

amorphous region or unreacted anhydride improving the hydrophilicity due to the presence of

oxygen species, or a combination thereof and improved the biodegradation rate. With the addition

of fibres, the water absorption and mass loss of grafted PHAs, perform poorly compared to non-

grafted samples 164,190,191,215,216,262, likely due to the available anhydride oxygen species being

bonded to fibres.

69

2.5.2.2 Surface Treatment

Surface treatment is an alternative method to improve fibre-matrix interfacial adhesion and the

overall properties by targeting the fibre/fillers themselves. In this regard, the polymer does not

undergo chain scission. Physical methods include treatments by plasma, electron irradiation and

surface roughening which can affect the hydrophilicity by removing hydroxide groups 263,264.

Chemical methods such as water washing or NaOH treatment can either wash away undesirab les

chemicals 265, or functionalize the fibre surface 263. NaOH treatment of fibres is a more effective

in improving the biocomposite properties when the matrix is more hydrophilic, such as in PHBV

compared to PE 83, mainly by substituting the hydrogen for Na+ to increase the polarity 266.

Furthermore, NaOH treatment can remove fibre lignin and hemicellulose, roughening the fibre to

maximize the surface area of fibres during biodegradation 263.

Silane treatment is a form of chemical modification of the fibre surface, which consumes hydroxyl

groups on cellulose as new chemical bonds are formed, and are reported to reduce the water

absorption in Sisal/PHBV composites 267. The removal of hydroxyl groups can also increase

surface roughness 268, but the method is more commonly used for non-polar polymers due to the

reduced fibre hydrophilicity 269.

2.6 PHA-Based Composites Biodegradation

Research on biodegradation of biocomposites following any ASTM standard is isn’t common

compared to virgin polymers. Several studies of PHA biodegradation have been completed with

non-bio-based fillers such as TiO2 192,270, carbon nanotubes, and organically modified clay claim

to slow the biodegradation of PHAs either by increasing composite carbon content 271, limit ing

segmental motion of high molecular chains or limit diffusion of water molecules into the bulk

sample 272. Natural based fillers provide a benefit from a sustainable point of view and the majority

are biodegradable in the appropriate environment. Natural rubber and other rubber constituents do

show some biodegradation behaviour but unfortunately slow the biodegradation of PHAs 273,274,

as a result of rubber biodegradation being a slow process requiring oxidation 275. PHA-based

70

biocomposites with natural fillers have the potential to reduce the cost and promote the

biodegradation of PHAs improving the water diffusion rate and maximizing water absorbed 133.

With the establishment that polysaccharide-based fillers can degrade when in composites, more

complex fillers have varying effects that can be considered beneficial for biodegradation. Fibres

such as bast when aged in water cause swelling of the cell walls, separating layers which can

improve the availability of enzymatic active sites 276. Long sisal fibres 5 mm in length are reported

to increase water absorption in PHBV/sisal composites, compared to 0.25 mm fibres. The fibre

loading is also positively correlated to water absorption 267. The absorbed moisture not only

separates layers, the fibre/matrix can debond, reducing the overall mechanical properties 256, but

in turn increase the capacity to absorb water. These factors attributed to the improved water

absorptivity and hydrophilicity of composites work in a similar way that hydrophilic plasticizers

improve PHA biodegradation 277. Furthermore, the physical structure of the fibre is important as

larger morphology negatively impact the biodegradation rate by reducing the surface-to-volume

ratio 278.

Biodegradation of biocomposites mainly begins around the interface of the matrix and the fibre

204, thus, any fibre treatment may affect the biodegradation rate. While MA grafting is reported to

have minimal effect 133, acetic anhydride and pyradine treatment can slightly inhib it

biodegradation but it remains unknown if the end % biodegradation will be affected, because no

such study was found at this time that was completed until a plateau 204.

The final attribute affecting the biodegradation of composites is the fibre composition. Natural

fibres contain a variety of glucose monomer units which can be broken up by cellulases, amylases

and cutinases, which are readily produced by microorganisms 98. Dewaxing of jute fibres before

composite fabrication can improve compostability of PHBV/Jute composites, by removing the

non-polar elements 279. As these natural fibres degrade, they also provide channels that allow water

and enzymes into the internal structure of the polymer matrix, thereby enhancing biodegradation

through increased surface area 280. Furthermore, biodegradation of polymers containing proteins

and lipids can biodegrade in soil, as the lipid and proteins provide a number of useful nutrients for

microorganism growth 281.

71

A major consideration of natural fibres are the limiting effects it has on conventiona l

biodegradation. Lignin is reported to slow microbial biodegradation and bio-assimilation due to

its complex structure and chemical formula, more suited towards fermentation processes 282.

Therefore, claiming biodegradability of biocomposites without an assessment of the individua l

components as a bare minimum, is a fallacy. Furthermore, natural fibres provide the more desirable

glucose as a carbon source which can slow biodegradation by glucose repression. It is still well

documented that PHA degradation is repressed in the presence of glucose and cellobiose 283,

However, once these specific carbon sources are degraded in a relative area, the PHA availability

will be maximized and its biodegradation will resume.

A second misinterpretation is the idea that cellulose derivatives will biodegrade, which is not

always correct. Cellulose acetate (CA), a derivative of cellulose, acts like a fibre when not

plasticized and can improve the biodegradation of PHA 284, provided its degree of substitution is

not high. Increased degree of substitution inhibits the biodegradation of CA, and beyond 2.5, it

shows no degradation at all under aerobic composting conditions 285. Cellulose acetate butyrate

(CAB) improved PHA elongation at break but reduced degradability 286 and, lignin-based PHB

composites reduced biodegradable performance 287. Thus, research of PHA-based natural fillers

and fibre biocomposites can prove invaluable in accelerating biodegradation and providing a more

sustainable approach in plastic research.

2.6.1 Composite Soil Biodegradation

Due to the relative ease with which soil biodegradation can be completed to obtain a general idea

of the effects of fibres, it is well populated with the effects of fibres on PHA biodegradation. The

most basic composites are made from starch or cellulose, both easily biodegradable by various

microorganisms. Increasing the content of starch improves the biodegradation of PHA 288. Studies

of more complex fibres such as wood, has also been studied in PHB, however, the improveme nt

is marginal 193. The complexity of fibre increases with the content of lignin; therefore, the fibre

type can have a significant effect on the PHA biodegradation improvement. Recent PHA

composite soil biodegradation studies are outlined in Table 2.5. With greater natural fibre and filler

addition, the soil biodegradation of every composite is reported to improve.

72

Table 2.5: PHA Composite Soil Biodegradation Studies from 2010-2020.

PHA (co-monomer %)/Composite

Protocol and Source

Conditions Form Days

Results Ref.

1 PHBV(1% HV)/ Wood

Flour (i) 80/20, (ii) 50/50

ASTM G160-

12, Subtropical Field

~20 °C,

7cm depth

1.6 mm

Plate

365 (i) 6.5%, (ii)

12.5% Mass Loss

221

2 PHBHx(3% Hx)/ Untreated Kenaf 70/30

Non-ASTM, Oil Palm Cultivation Soil

30 °C, 81% Relative

Humidity

3 mm Plate

42 13% Mass Loss 220

3 PHBHx(3% Hx)/ Alkali and Acid Kenaf

70/30

Non-ASTM, Oil Palm

Cultivation Soil

30 °C, 81%

Relative Humidity

3 mm Plate

42 7.5% Mass Loss 220

4 PHA/Palm Fibre (i) 80/20, (ii) 60/40

Non-ASTM, Farmland

Topsoil

30-40% Moisture

(w.b.)

Film 60 (i) 72%, (ii) 90% Mass Loss

191

5 PHA-g-MA/Silane Treated Palm Fibre (i) 80/20, (ii) 60/40

Non-ASTM, Farmland Topsoil

30-40% Moisture

(w.b.)


191

6 PHA/Marine Algae Powder (i) 90/10, (ii)

80/20

Non-ASTM, Greenhouse

25 °C, 35%

Moisture

(w.b.)

Powder 120 (i) 70%, (ii) 88% Mass Loss

216

7 PHA-g-AA/Silane

Marine Algae Powder (i) 90/10, (ii) 80/20

Non-ASTM,

Greenhouse

25 °C,

35% Moisture

(w.b.)

Powder 120 (i) 61%, (ii)

78% Mass Loss

216

8 PHB/Wood Fibre 80/20

ISO 17556, Forest Soil with Garden Soil

20 °C, 40-60% Moisture

(w.b.)

Pellet 195 60% Biodegradation

193

9 PHA/Tea Plant Fibre (i) 80/20, (ii) 60/40

Non-ASTM, Farmland

Topsoil

35% Moisture

(w.b.)


190

10 PHA-g-MA/Silane Treated Tea Plant Fibre (i) 80/20, (ii)

60/40


35% Moisture

(w.b.)


190

11 PHA/Rice Husk (i)

80/20, (ii) 60/40

Non-ASTM,

Farmland Topsoil

35%

Moisture

(w.b.)

Plate 60 (i) 82%, (ii)

95% Mass Loss

215

73

12 PHA-g-AA/Rice Husk (i) 80/20, (ii) 60/40


35% Moisture

(w.b.)

Plate 60 (i) 75%, (ii) 90% Mass Loss

215

2.6.1.1 Effect of Natural Fibre/Filler Type

Soil biodegradation of PHA biocomposites has been completed with several fibres and fillers

including starch, soy, lignin, flours, wheat straw etc. Starch based granules are a separate subset

among natural fillers due to the alpha repeating units of glucose, providing a direct source of

functional carbon in an optimal form with minimal degradation. Increasing the starch content from

10 to 30% increased the % degradation of PHA by 34%, indicating there are synergistic benefit

with the presence of fibres 288. Wei et al. 289 reported PHB/potato peel waste composite films where

the potato peel waste component degraded in the early stages through analysis of the melt

temperature, which are the result of potato peel waste being composed mainly of starch (66%)

despite containing some cellulose, hemicellulose and lignin 290.

The complex structure of lignin is not easily degraded in soil biodegradation and showed evidence

of no biodegradation compared to other natural fillers when in a PHA matrix in an aerobic

environment 288. Although lignin is reported to act as channels for degradation of the polymer

matrix in anaerobic environments 291, it severely retarded degradation in anaerobic environments

181. Wheat straw contains 16-25% lignin 292, which is expected to inhibit the degradation of PHAs.

Avella et al. 173 reported PHBV/wheat straw 70/30 samples degraded by 23% in 180 days in garden

soil, but no improvement in the rate of biodegradation was found compared to PHBV. PHA/wood

flour 50/50 composites under natural and laboratory conditions is reported to show wood flour

degradation improvements although the two studies vary from 12.5% in 365 days to 50% in 84

days respectively, which can be attributed to the higher surface area and moisture in laboratory

studies 164,221. Furthermore, depending on the type of wood flour the lignin content can vary from

21-34% 293.

The presence of lignin is still not enough to determine if the biodegradation rate will be impacted

and to what extent. PHA/rice husk 80/20 biocomposites improved the water absorption of PHA

74

and increased the % mass loss in farmland topsoil from 33 to 75% in 60 days 215, despite rice husk

containing 25-30% lignin. However, rice husk also contains 50% cellulose and 20% silica 294, the

former being easily degradable and contains only glucose unlike hemicellulose and lignin.

Hemicellulose does provide benefits, being the amorphous fraction of the fibre, it is more easily

hydrolysed. Peach palm fibre contains a significant amount of hemicellulose which is composed

of a number of other hexose and pentose monomers besides glucose 295, and 0-25% weight ratios

in PHBV showed a slight improvement in physical disintegration 218. Abaca fibre contains 66%

cellulose, 24% hemicellulose and 12% lignin, giving a balance between the cellulose and

hemicellulose, with little lignin 296, and 10% abaca fibre improves the degradation of PHBV from

30% to 50% in 180 days in regularly watered gardening soil at 25-30 °C 204. Kenaf fibre has

comparable cellulose and lignin content to that of abaca fibre with 13% hemicellulose 247, and

PHBHx/kenaf 70/30 composites improve the degradation from 5.5 to 13% in 42 days 220 (Table

2.5. Entry 2). Therefore, it is not only the ratio of cellulose, hemicellulose and lignin that plays a

role in the biodegradation improvement of PHA, but also the composition of the hemicellulose

monomers (i.e. hexose, pentose glucose etc.).

Natural fillers such as grains include large amounts of protein besides, cellulose, hemicellulose

and lignin. Protein provides a nitrogen source, which after processing can be more readily available

after protein degradation. PHA/soy 67/33 composites degraded by 89% in 168 days, exceeding

similar ratios of PHA/starch composites 288. The nitrogen can be utilized by microorganisms for

proteins and cell growth. The proteins (with a variety of essential amino acids), oils and cellulos ic

fibre found in DDGS 297 can also provide suitable nutrients to allow growth of microorganisms

and are significantly cheaper. The composition of DDGS in PHA/DDGS composites have allowed

for comparable degradation to that of PHA/starch composites of similar weight ratios 288.

Additionally, Madbouly et al. 219 reported 10% DDGS increased the biodegradation rate of PHA

by 6 times. However the study was not completed until a plateau, and also utilized clarion loam

soil which is known to have very good cellulolytic activity 124.

Other natural PHA-based composites include PHA/marine algae powder, which performed poorly

compared to PHA/rice husk under similar weight ratios and study conditions 215,216 (Table 2.5.

75

Entry 6 and 11). Marine algae powder can have 15-66% polysaccharides (starch based or similar),

making it unknown what was affecting the biodegradation rate 298.

2.6.1.2 Effect of Fibre Treatment

Fibre treatment can be used to improve the surface characteristics for not only the interfac ia l

adhesion, but also the interaction between water/enzymes and the fibres. The most basic fibre

treatment studied in soil biodegradation is alkali or acid treatment. Acetic acid and pyradine treated

abaca fibre slightly improved the water absorption but failed to improve the biodegradation any

farther than PHBV/abaca fibre composites 204. Joyyi et al. 220 reported alkali and acid treated kenaf

fibres in PHBHx/kenaf 70/30 biocomposites had no effect on water absorption and reduced the

degradation (Table 2.5. Entry 3). The alkali treatment is indicated to remove the hemicellulose and

lignin 299, which would result in a reduced amorphous fraction that may limit its susceptibility to

enzymatic and hydrolytic attack.

Silane treatment of fibre surfaces was been studied several types of fibre by Wu et al. 20% loading

of marine algae powder 216, palm fibre 191 or tea plant fibre 190 increased the soil biodegradation

rate of PHA by 100% (Table 2.5. Entry 5, 7 and 10). However, with treatment of the fibres with

tetraethyl orthosilicate or tetraethoxysilane, the biodegradation rate of the composites decreased

by approximately 10%. The crosslinking effect upon the fibre can interfere with the enzyme

activity, in addition to increasing the molecular weight. Furthermore, the ethyl groups on the silane

agent are hydrophobic which can reduce the water absorptivity of the fibres. Acetylated or silane

treated fibres similarly reduced the physical degradation in PBS-based composites in soil 300 and

composting 301.

Other forms of fibre treatment completed in composite soil biodegradation are regenerated

cellulose (lyocell) and peat (decayed matter) which represent special attributes that make them

suitable for low release of fertilizers. PHBV/lyocell 60/40 biocomposites were studied under soil

biodegradation with 80% humidity, but showed no improvement in biodegradation compared to

PHBV. Shibata et al. 213 reported PHBV matrix coated the lyocell and did not allow the advantages

of the filler to enhance the degradation of the composite. These characteristics are not seen in

76

PBS/lyocell and PLA/lyocell composites and can be attributed to the hydrophobicity of PHAs. In

such cases fertilizer encapsulated by PHA can be released only once degradation of PHA proceeds

to a certain extent. Peat is the decayed form of organic matter, and compared to wood flour it has

a lower moisture absorption but still improves the biodegradation rate of the composite to a greater

degree 302. Peat also has the added benefit of providing beneficial nutrients to plant growth.

The main applications for soil biodegradation of PHA biocomposites is modulation of

encapsulated nutrients, fertilizers and other compounds. PHA based composites with oil palm

fibres have already been studied to create slow release fertilizers in the agricultural industry 303.

This solution can be applied to several PHA biocomposites if PHA biodegradation needs to be

accelerated and low-cost natural fillers.

2.6.2 Composite Composting

PHA composite composting, is important due to the societal focus on implementing composting

services. PHA composites hold the potential to fulfil a rapid degradative role that PLA is unable

to achieve. Recent PHA composite composting studies are outlined in Table 2.6. Composting

studies on three forms of PHA/cellulose composites have been completed. Under industr ia l

composting conditions (no standard), PHB is reported to degrade by 50% in 84 days, and with 10-

30% lyocell loadings the biodegradation slightly improves to 55-68% in 84 days 230 (Table 2.6.

Entry 7). Sanchez-Safont et al. 304 reported similar findings in PHBV/cellulose composites where

there was no significant improvement in the degradation rate or extent with cellulose incorporation

under ISO 20200 in 54 days (Table 2.6. Entry 9). 5% Cellulose nanocrystals in PLA/PHB 75/25

blends halved the degradation period from 21 days to 10 days which can be attributed to the

increased surface area nano sized particles have. However, 5% surfactant was utilized to modify

the nano crystal but delayed onset of degradation by 4 days 305. Given that cellulose has minimal

effect on the compostability of PHAs, it can be attributed to the cellulose crystalline structure.

Furthermore, cellulose may situate itself inside the PHA matrix, minimizing microorganism

exposure in the initial stages of composting.

77

Under natural composting conditions, PHBV/starch composites showed remarkable improvement

in degradation rate, increasing PHBV(12% HV) degradation from 7-25% and 49%, with 30 and

50% starch loadings respectively. The increased starch content is also reported to increase the

PHBV degradation rate in the composite 306. Starch is well known to have an amylose and an

amylopectin fraction, the former being amorphous in nature which can reduce the crystallinity of

the composite. Furthermore, starch is hydrophilic in nature, benefitting enzymatic catalysed

hydrolytic degradation and is further reflected by its faster rate of soil biodegradation compared to

cellulose 170. These attributes are also seen in hemicellulose, but lignin is reported to reduce the

crystallinity, however, it does reduce the degradation extent 149. Lignin composites perform poorly

compared to starch in composting conditions with both a PHA and a PLA matrix 307. Therefore, a

crystallinity reduction is not adequate to improve the biodegradation rate and some hydrophilic

functions as well as a relative ease of degradation is required. This is further illustrated by

PHBV/wheat straw composites which reduced the crystallinity but had a negative impact on the

degradation rate in compost 173. The main cause is attributed to the high lignin (16-25%) and low

cellulose 292.

Aside from starch, the neither cellulose nor lignin benefit PHA composting in the high ratios.

Gunning et al. studied PHB based jute, hemp and lyocell composites. With 30% loadings of the

natural filler, jute increased % degradation from 50% to 85% while hemp and lyocell increased it

to 68% 230 (Table 2.6. Entry 6-8). The mass rate improvement can be attributed to the slightly

lower cellulose and higher hemicellulose content in jute fibre.

Other considerations found in composting is the presence of protein which reflects the effect seen

in soil biodegradation. PHA Soy 67/33 and PHA/DDGS 80/20 were both reported to have 100%

degradation in 84 days, unlike PHA/starch which was at 57% 307 (Table 2.6. Entry 2-4). Both

natural fillers contain nitrogen sources for microbial growth. The effect of DDGS is also seen in

PBAT, which made the composite degradation initial stages comparable to cellulose 308. DDGS

composition is approximately 19% cellulose, 17% hemicellulose, 5% starch an 30% protein 265,

making it an amorphous, hydrophilic, nitrogen containing source suitable for biodegradable

composites.

78

Table 2.6: PHA Composite Composting Studies from 2010-2020.



1 PHBV(20% HV)/Lignin

80/20

ASTM D5338,

Mature Compost

Film 60 85%

Biodegradation

149

2 PHA/Soy 67/33 Non-ASTM, Home Composting (21-61 °C)

Small Pot

84 100% Mass Loss

307

3 PHA/DDGS 80/20 100% Mass Loss

4 PHA/Starch 90/10 57% Mass Loss

5 PHA/Lignin 80/20 21% Mass Loss

6 PHB/Jute (i) 90/10, (ii) 80/20, (iii) 70/30

Non-ASTM, Home Composting (34-66 °C)

Tensile Sample

84 (i) 65%, (ii) 68%, (iii) 85% Mass Loss

230

7 PHB/Lyocell (i) 90/10, (ii) 80/20, (iii) 70/30

Non-ASTM, Home Composting (34-66

°C)

Tensile Sample

84 (i) 55%, (ii) 58%, (iii) 68%

Mass Loss

230

8 PHB/Hemp (i) 90/10, (ii) 80/20, (iii) 70/30

Non-ASTM, Home Composting (34-66

°C)

Tensile Sample

84 (i) 58%, (ii) 62%, (iii) 68%

Mass Loss

230

9 PHBV(3% HV)/Cellulose 97/3, 90/10, 75/25, 55/45

ISO 20200, Mature Compost

Plate 47 100% Mass Loss

304

10 PHB/Wood Fibre 80/20 ISO 14855, Organic

Municipal Solid Waste

Pellet 78 95%

Biodegradation

193

11 PHBV(5% HV)/Wheat

Straw (i) 90/10, (ii) 80/20, (iii) 70/30

Modified ASTM

D5338, Mature Compost

Film 48 (i) 55%, (ii)

60%, (iii) 65% Biodegradation

173

2.6.3 Composite Marine Biodegradation

Although PHA are well known for their marine biodegradable properties, however, the research

into the effect of natural fillers in the PHA matrix remains relatively unexplored compared to soil

biodegradation and composting (Table 2.7). The ideal representation of marine degradation of

PHA composites is PHBV/corn starch which showed rare biphasic degradation which also follo ws

79

the ideal bacterial growth curve 154. Imam et al. reported the presence of starch also improved the

degradation rate of PHBV as the composite biodegradation exceeded the combination of the two

constituents. There is evidence enzymes may penetrate the PHA wall to degrade the filler and

adherence of filler to polymer matrix is critical to whether the filler can be degraded effectively 140

(Table 2.7. Entry 1). Biphasic degradation is disadvantageous because the overall composite

degradation rate may remain unchanged. Ramsay et al. 237 reported 25 and 50% wheat starch

reduced the PHBV degradation time from 32 days to 21 and 8 days respectively. The significant

improvement in PHA biodegradation is the result of improved water uptake which caused fibre

swelling and some mechanical strain to assist in degradation action 309 (Table 2.7. Entry 3). A

second advantage of fibre incorporation into PHA composites for marine biodegradation is the

enhancement of biofilm growth 254, which is more common in stable or static environments in soil

or compost. Therefore, natural fillers addition into PHAs improve the marine biodegradation by

allowing enzyme permeation into the amorphous fractions. However, the literature available on

composite marine biodegradation is limited, and it remains unclear whether the improved enzyme

motility is a characteristic of starch or the increased water content.

Table 2.7: PHA Composite Marine Biodegradation Studies.


Protocol and Source

Conditions Form Days Results Ref.

1 PHBV(12%

HV)/Starch (i) 70/30, (ii) 50/50

Non-ASTM,

Tropical Coastal Water

26-32 °C Tensile

Sample

365 (i) 80%, (ii)

100% Mass Loss

140

2 PHBV(19%

HV)/Starch (i) 75/25, (ii) 50/50

Non-ASTM,

Anaerobic Digester Water

30 °C Film 150

µm

(i)

21, (ii) 8

100% Mass Loss 237

3 PHBV(19%

HV)/Starch (i) 75/25, (ii) 50/50

Non-ASTM,

Anaerobic Digester Water

30 °C Film 800

µm

(i)

32, (i) 21

(i) 85%, (ii)

100% Mass Loss

237

4 PHBV(5% HV) composite/Seagra

ss (i) 90/10, (ii) 80/20

Modified ASTM D6691,

Seawater and Sediment

30 °C Pellets 216 (i) 20%, (ii) 27% Biodegradation

254

5 PHBV(5% HV)

composite/Seagrass 80/20

Non-ASTM,

Mariculture Centre

12-27 °C Tensile

Sample

365 23% Mass Loss 254

80

2.6.4 Conclusions of Composite Biodegradation

Most factors that positively impact PHA biodegradation are clearly carried over to PHA-based

composite biodegradation. The addition of most fibres, no matter the type, improve the

biodegradation. Starch is the most beneficial fibre to improve PHA biodegradation due to its

hydrophilicity, amorphousness and ease of biodegradation in soil and marine environments.

Natural fibres benefit PHA biodegradation when the cellulose and hemicellulose are in balanced

ratios as hemicellulose reduces crystallinity, while cellulose and hemicellulose to a lesser extent

are both readily biodegradable. PHA/Jute composites degrade faster than PHA/lyocell composites.

Lignin, inhibits biodegradation, despite reducing crystallinity due to its complex structure making

degradation difficult in all aerobic and anaerobic biodegradation environments (excluding

fermentation). Fillers such as DDGS and Soy which contain protein provide a nitrogen source

which benefit microbial growth and further enhance biodegradation rates in both soil and

composting conditions.

Treatment of fibres by acids, alkalines, or silanes as per the literature found in this review had no

improvement on the water absorption and reduced the degradation in PHAs. The processes either

removed the hemicellulose fraction, increasing crystallinity, or crosslinked the fibres, inhibit ing

the enzymatic functions. Grafting of the PHA with anhydrides did improve the extent of

degradation - however, with the addition of fibres, the grafted PHAs performed poorly due to

crosslinked fibres inhibiting enzymatic activity. Other forms of structural treatment such as

cellulose regeneration (lyocell) gave no improvement, and the use of decaying organic matter

improved the biodegradation of PHAs.

2.7 Conclusion

For a sustainable circular economy that mitigates the negative effect on the environment and the

eco-system of conventional plastics, research and development into bio-based biodegradable

polymers is imperative. Not only can bio-based biodegradable polymers reduce greenhouse gas

emissions, but they can combat mismanaged waste that leaks into the environment, where human

81

intervention is ineffective. And depending on the end-of-life, there is significant employment

generation potential for value-added compost, for example. To implement these biodegradable

polymers a deep understanding of the environments and their corresponding standards is

necessary. Soil and marine environments are representative of a large pool where mismanaged

waste ends up, such that studies in natural biodegradation environments can supplement

development in improvements of biodegradable polymers.

PHAs are considered the most readily biodegradable polymer among many (i.e. PLA, PBS, PBAT

etc.), supported by literature, in aerobic environments (soil, compost and marine), and showing

promising biodegradable behaviour in anaerobic (sewage sludge, digesters and landfills)

environments. PHAs benefit from biotic degradation by several types of bacteria and fungi

enzymes. Furthermore, the surface area can be maximized and crystallinity lowered based on

different processing techniques or longer chain PHAs to optimize the biodegradation performance.

The characteristics all improve the permeation of moisture and enzyme interaction that accelerate

the enzymatic catalysed hydrolysis. Furthermore, PHAs degradation products are easily

assimilated into usable products for microbial growth.

However, the addition of certain additives (chain extenders) and other biodegradable/non-

biodegradable polymers that are used to improve its properties can be detrimental to its

biodegradable performance. The negative effects of Joncryl, antifouling agents and non-

degradable or hydrophobic plasticizers inhibit the onset and/or extent of PHA biodegradation in

aerobic and anaerobic environments. Although these attributes are beneficial for biodegradation

modulation in agricultural industries, it is not desirable for single use plastic consumer waste.

Blending PHAs with PLA, PBAT etc. all inhibit the biodegradation of PHAs in most

environments, except compost, where PLA does outperform PHAs.

To further improve the biodegradation of PHAs, incorporation of natural fibres and fillers can

accelerate the rate of biodegradation based on the composition of fibres, while inorganic fillers

either inhibit biodegradation or do not degrade at all. Natural fibres high in hemicellulose and

cellulose benefit from improved hydrophilicity and eased biodegradation respectively. Fillers with

high lignin and cellulose with low hemicellulose can inhibit the biodegradation of PHAs due to

82

their complexity and low hydrophilicity respectively. Starch based fillers provide high

hydrophilicity and eased biodegradation, allowing PHA biodegradation to significantly improve.

Furthermore, by incorporating fillers with proteinaceous material such as DDGS and soy, the

biodegradation of PHAs can improve beyond starch and natural fillers without. However, the

research in the area of PHA biocomposite biodegradation is severely limited in marine

environments, where a significant mismanaged plastic waste is found.

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121

Chapter 3: Sustainable PHBV/Cellulose Acetate Blends: Effect of

Chain Extender and Plasticizer

Abstract

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and cellulose acetate (CA) were blended

in the presence of a plasticizer, i.e., triethyl citrate (TEC), and a chain extender, i.e., poly(styrene-

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temperature. Due to processing at high temperature (210-220 °C), significant porosity was

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improved by 110% as compared to virgin PHBV. The addition of CE had no effect on the

mechanical properties but did make the PHBV/pCA blends morphologically uniform.

122

3.1 Introduction

The use of plastic packaging materials, especially single use plastics produced from petroleum-

based sources is a growing environmental concern. In fact, a large proportion of the materials end

up in landfill or in the environment, taking more than a lifetime to degrade. Nearly 36 % of plastic

was utilized for single use applications in 2017; of that, approximately 14 % was recycled, while

the rest was incinerated, landfilled or remains in the environment 1. Similarly, light weight and

littered plastics migrate to the ocean, take thousands of years to degrade and significantly increases

the removal difficulty, which creates problems for marine life 1. Polyhydroxyalkonoates (PHAs),

and more specifically poly(3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), is a potential

resource to mitigate further damage to the environment. It is currently sourced from natural

renewable fermentation sources and is a 100% biodegradable and compostable polymer 2, making

it an attractive sustainable alternative. It is worth mentioning that PHBV is one of the few

biopolymers that is marine biodegradable 3, soil biodegradable 4 and compostable under

appropriate conditions 5.

PHAs are bacterial polyesters, synthesized by prokaryotic organisms from several carbon sources,

such as agricultural and industrial waste, and other mixed carbon sources 6. With inherent

biodegradable properties, PHAs form a closed loop, sustainable cycle “from cradle-to-grave”, that

minimizes their impact on the environment. PHBV, for example, is sourced from natural

renewable fermentation sources 2. PHBV can be classified as a very brittle material with a narrow

processing window 7, especially with low hydroxyvalerate content (2 to 5%), having a tensile

modulus of 3.2 GPa with an elongation at break of 1.4 % 8, comparable to PLA. However, petro-

based polymers such as polypropylene have an elongation at break above 50 % 9.

Despite this limitation, PHAs are commonly used in biomedical applications. However, they must

be free of organic impurities from production, such as carbohydrates or proteins, that can activate

the immune system in humans 10. In packaging applications, PHBV is more suited to specialty

packaging due to higher production costs. However, the relative inertness compared to other

plastics make it suitable for active and passive food packaging applications 11.

123

For flexible packaging applications, the thermo-mechanical properties and the production cost are

of far more concern. PHBV, being more ductile than PHB, is still susceptible to thermal

degradation due to the high shear, temperature and residence time during processing, significantly

limiting its applications 7. A common approach to overcome these limitations is to blend PHBV

with other biodegradable polymers to increase the processing window and minimize final product

costs 12, while optimizing the mechanical and thermal properties.

Cellulose, a common building block of organic life, is a highly crystalline polysaccharide which

is insoluble in all organic solvents 13. Thus, it is usually derivatized into cellulose esters to improve

processability 14. Common cellulose acetate esters are cellulose acetate (CA), cellulose acetate

butyrate (CAB) and cellulose acetate propionate (CAP). CA esters are commercially produced by

substituting hydroxyl groups with acetyl, butyryl and propionyl groups with a degree of

substitution (DS) in the range of 1.7 to 3.0 14. Among the CA esters, CA is one of the most used

cellulose derivatives 15.

Miscibility plays an important role in polymer blends, allowing for a homogenous mixture during

processing and optimized thermo-mechanical properties. However, thermodynamica lly

immiscible blends reflect distinct phase separation during melt blending 16, and decimated

mechanical properties, which can only be mitigated by compatibilizing agents 17.

Through solvent casting, PHBV and cellulose triacetate, CA with a DS of 3.0, were reported to be

miscible and have improved tensile properties and acid catalyzed hydrolytic degradation rate 18.

However, the research literature available on CA blends with PHB or PHBV was limited, hence,

PHB and PHBV blends with other CA esters were investigated. A number of other studies reported

PHB and PHBV to be partially or completely miscible over the entire range with other cellulose

esters, including CAB and CAP 19–22.

The DS effect in various CA derivatives significantly impacts their utility by changing their

properties. Completely substituted CA has a DS of 3. According to Gardner et al., as the DS

increases, biodegradability decreases or is entirely halted, but CA with a maximum DS of 2.5

degrades to CO2 23. Other CA esters, such as CAB, are very stable polymers, and significantly less

124

susceptible to biodegradation 24. However, CA is commonly plasticized due to its poor thermal

stability at melt temperature to improve its processability 14.

Among the available plasticizers (e.g., polyethylene glycol, tributyl citrate etc.), triethyl citrate

(TEC) has been used in both PHBV and CA, and is a non-toxic, bio-based plasticizer that is safe

for use in food packaging, making it an attractive option to plasticize cellulose acetate 25–27.

However, the mechanical properties of CA vary significantly depending on the polymer to

plasticizer ratio. CA with 20-40% TEC content has better mechanical properties when processed

at 180 °C or lower temperature, with high shear conditions such as extrusion at 100 rpm 25.

Through literature, the optimal ratio of CA to TEC plasticizer was found to be 25 % with a melt

compounding temperature of 200-210 °C 28.

The literature survey found that research on PHBV and CA ester blends is limited, mostly

containing PHBV and CAB or CAP blends. The processes involved were solvent casting, melt

mixing followed by compression moulding and injection moulding 20–22,29. PHBV/CAP blends

indicated improved thermal stability and miscibility due to good phase interaction 29. PHBV/CAB

blends were evaluated to be thermally miscible with <50% PHBV content 21, however, El-Shafee

et al. reported PHBV to be entirely miscible with CAB 30. Buchanan et al. reported improved tear

strength positively correlated to PHBV, and an elongation of 106 % for PHBV/CAB (50/50)

compression moulded samples 20. However, the limits of the literature survey are reflected by the

lack of mechanical properties for PHBV and CAB or CAP blends in the sources cited above.

PHBV and completely substituted CA (DS = 3) were blended and reported to be miscible to some

extent, but only through solvent casting, and only one blend ratio (36/64) was reported. The

Young’s modulus and tensile strength were improved by 12.7 and 36%, respectively, for

PHBV/CA (36/64) blends 18. Melt extrusion or other processing techniques of PHBV/CA blends

were not found in any form with other DS for CA.

The expected outcome of blending PHBV and CA together is to incorporate the functiona l

properties of CA into PHBV’s degradation processes. Cellulose triacetate was reported to improve

degradation of PHBV in acidic conditions. Cellulose triacetate increases the water uptake and

125

water vapour permeability 18, therefore, it can potentially increase the water-PHBV interfac ia l

surface area and increase biodegradability in marine water.

Furthermore, it has already been researched that CA plasticized with 25% TEC produced the

optimal effect, thus, the processing temperature of PHBV must be considered. PHBV is sensitive

to the processing temperature, however, a chain extender such as Joncryl (ADR-4368 S) at 0.25 to

1% is reported to effectively increase the viscosity and extend the molecular chain length of PHBV,

effectively rebuilding the molecular weight 31. However, no improvement in the mechanica l

properties was reported 32.

In this research work, PHBV and CA plasticized by TEC for applications in biodegradable flexib le

packaging were melt compounded and extruded to determine the miscibility for the first time.

Furthermore, as per our literature review, this is the first study of a sustainable blend using PHBV

and CA in extrusion. The objectives of this study were to assess the miscibility of different blend

ratios of PHBV/CA by characterizing the thermal and morphological properties. The mechanica l,

physical and thermal properties of PHBV/CA blends were thoroughly investigated to optimize the

performance. Additionally, the effect of a chain extender on the mechanical, thermal and

morphological properties of PHBV/CA blends was studied and the effect of processing

temperature and plasticizer content are discussed based on the mechanical and thermal properties.

3.2 Materials and Methods

3.2.1 Materials

PHBV pellets, CA powder and TEC as a plasticizer were the main materials used in this research.

The PHBV pellets, with the tradename ENMAT Y1000P, were obtained from Tianan Biologica l

Materials Co. Ltd and are reported to have 1-5 mol% HV content. The CA powder (CA-398-30,

acetyl content: 39.8 w.t.%, hydroxyl content: 3.5 w.t.%) was purchased from Chempoint. USA.

TEC was purchased from Sigma-Aldrich with the product code W308307-1KG-K. Poly(styrene-

acrylic-co-glycidyl methacrylate (SA-GMA) (Joncryl ADR-4368C, BASF, Germany), was used

as a chain extender. All the polymers and chemicals were used without any further purificat ion.

Blends including the chain extender SA-GMA (CE) were prepared by adding CE to PHBV at 0.3

126

percent hundred rubber (phr) of the total blend mass. Following CE addition, blends were prepared

and processed as described below.

3.2.2 Preparation of Plasticized Cellulose Acetate

Prior to processing, both PHBV and CA powder were vacuum dried in an oven at 80 °C overnight.

The moisture content of each polymer was measured using a moisture analyzer (Sartorius MA37)

prior to powder plasticization. The CA powder, when being plasticized, was mechanically mixed

with the requisite TEC% and left at room temperature overnight. pCA denotes the plasticized CA

powder with 25 % TEC.

3.2.3 Melt Extrusion Followed by Injection Moulding

The melt extrusion was performed in a DSM Explore twin screw batch extruder (DSM research,

Netherlands). The processing temperature ranged from 200-210 °C or 210-220 °C depending on

the blend composition, unless otherwise stated. Furthermore, the retention time and screw speed

were maintained at 2 mins and 100 rpm, respectively. PHBV was not injection mouldable above

temperatures of 180 °C due to an increased MFI and PHBV liquidation, making the injection

molded parts incomplete in the DSM, which was why blends with high PHBV content were

processed at 200-210 °C. CA and TEC blends were the only formulations with 6 min retention

time as opposed to 2 min. CA and TEC blends were not processable at high temperatures or shorter

times due to poor melt flow making injection moulding difficult. The molten polymer blends were

injected into molds maintained at 30 °C using a micro-injector at a melt temperature of 200-220

°C, with the fill, hold and pack pressures all set to 10 bar with each having a 6 s duration. The

blend compositions are presented in Table 3.1.

Table 3.1. PHBV/CA blend compositions.

Notation PHBV

(w.t.%)

CA

(w.t.%)

TEC

(w.t.%)

CE

(phr)

Processing

Temp. (°C)

PHBV 100 0 0 0 180

PHBV/pCA (70/30) 70 22.5 7.5 0 200-210

PHBV/pCA (50/50) 50 37.5 12.5 0 210-220

PHBV/pCA (30/70) 30 52.5 17.5 0 210-220

pCA 0 75 25 0 200-210

127

PHBV/pCA/CE (70/30/0.3) 70 22.5 7.5 0.3 200-210

PHBV/pCA/CE (50/50/0.3) 50 37.5 12.5 0.3 210-220

PHBV/pCA/CE (30/70/0.3) 30 52.5 17.5 0.3 210-220

3.2.4 Solubility Calculations

The solubility parameters were calculated by using two methods i.e., the Hoy method and the

Hoftyzer-Van Krevelen method, which use the groups of a polymer chain (i.e. -CH2, -CH3, -OH

etc.) found in PHBV, CA and TEC to derive the dispersive, polar and hydrogen bonding cohesive

forces for calculating the individual solubility components. The square root of the sum of the

squares of the polar (δp), dispersive (δd) and hydrogen (δh) bonding forces gives an overall

solubility (δ) for each constituent of the blend. The basic assumptions behind the solubility

parameters are that the dispersive, polar and hydrogen forces make up the cohesive force of a

molecule and can, therefore, be equated to the solubility parameter. Furthermore, the interactions

between two such as chemical reactions and during melt mixing is not well represented by Hansen

solubility parameters and the overall calculated solubility parameter 48. The molecular weight (Mw)

of PHBV Y1000P, CA (CA 398-30) and TEC are 240,000 34, 143,000 49 and 276.283 g/mol.

3.2.5 Differential Scanning Calorimetry (DSC)

All blends and virgin polymers were analyzed by differential scanning calorimetry (Q200, TA

instruments, Delaware, USA). Each sample (5-10 mg) was subjected to a heating, cooling and

heating cycle under a nitrogen atmosphere with a flow rate of 50 mL/min. In the first heating part

of the cycle, the rate was 10 °C/min from -50 to 200 °C followed by isothermal conditions for 3

min to erase the thermal history of the polymer blend. The first cooling part was at a rate of 5

°C/min to -50 °C and equilibrated for 3 min. The second heating part was at a rate of 10 °C/min to

240 °C. In virgin PHBV samples, the second heating part of the cycle was halted at 200 °C to

avoid the degradation of PHBV. The first cooling part was used to observe the enthalpy of polymer

crystallization (ΔHc) and the peak crystallization temperature (Tc). The second heating part was

used to determine the melting temperature (Tm) and the enthalpy of melting (ΔHm). The %

crystallinity (𝑋𝐶) of the PHBV in polymer blends was calculated using Equation 3.1 50.

128

𝑋𝐶 = (∆𝐻𝑚

∆𝐻𝑚0 × 𝑤𝑓

) × 100 %

Equation 3.1

Where, ∆𝐻𝑚 denotes the overall enthalpy of the melting peak(s), ∆𝐻𝑚0 denotes the theoretica l

enthalpy of 100 % crystalline PHBV, which is reported to be 109 J/g 51, and 𝑤𝑓 is the weight

fraction of PHBV in the sample 50. Two replicates were completed for each sample.

3.2.6 Thermogravimetric Analysis (TGA)

Virgin PHBV and CA, in addition to the TEC and fabricated blends, were analyzed using a

thermogravimetric analyser (Q500, TA instruments, Delaware, USA). Each sample (15-20 mg)

was subjected to heating at a rate of 10 °C/min from room temperature (~21 °C) to 600 °C in a

nitrogen enriched environment with a purge and balance flow rate of 40 and 60 ml/min,

respectively. The derivative thermogravimetric analysis (DTGA) peaks were taken as the peak

degradation rate temperatures for each blend. Two replicates were completed for each sample.

3.2.7 Heat Deflection Temperature (HDT)

The heat deflection temperature (HDT) was analyzed for melt extruded virgin polymers and their

blends using a dynamic mechanical analyser (Q800 from TA instruments, Delaware, USA) under

3-point bending. In accordance with ASTM D648, 0.455 MPa was applied to impact bars and the

strain was measured as the temperature increased. Starting from 30 °C, the heating rate was 2

°C/min until the strain exceeded 0.22%. The average of the temperature at the displacement of 250

µm for the two samples was taken as the HDT.

3.2.8 Dynamic Mechanical Analysis (DMA)

The storage modulus (E’), loss modulus (E’’) and the loss tangent (tan δ=E’’/E’) of all fabricated

specimens were measured using the dynamic mechanical analyser (Q800 from TA instruments,

Delaware, USA) under multifrequency strain with a dual cantilever. The samples were heated at 3

°C/min from -50 °C to 140 °C or when the drive force approached 0 Newtons. The applied

129

frequency for all samples was 1 Hz with an amplitude of 15 µm. The loss tangent peak was utilized

to determine the glass transition temperature (Tg) of the samples. TA analysis software was utilized

to analyze the results. Two replicates were completed for each sample.

3.2.9 Tensile and Flexural Properties

Five tensile and flexural samples were conditioned before analysis for 48 h at 21 °C with a relative

humidity of 50 %. The tensile and flexural strength were measured using an Instron 3382 Universa l

Testing Machine (Massachusetts, USA). In accordance with ASTM D638, type IV tensile bars

were tested at room temperature and humidity conditions with a rate of 5 mm/min. In accordance

with ASTM D790, flexural bars were tested over a 52 mm span in a three-point bend configura t ion

with a rate of 14 mm/min. Bluehill software was utilized to process the test results.

3.2.10 Notched IZOD Impact Strength

The impact strength of samples was measured utilizing a Zwick Roell HIT25P impact tester (Ulm,

Germany) in accordance with ASTM D256. Each sample was notched immediately after

processing. The average of six replicates was taken as the notched IZOD impact strength.

3.2.11 Density

Density of samples was measured using an Electronic Densimeter MD 300S (Florida, USA). Three

flexural bars were utilized for each measurement and the average was taken as the density.

3.2.12 Scanning Electron Microscopy (SEM)

SEM micrographs of the impact fractured sample break surfaces were obtained using a Phenom

ProX Desktop from Phenom-World BV (Eindhoven, Netherlands). Charging was minimized by

using a Cressington Sputter Coater 108 (Watford, England) to apply a thin gold coating on the

fractured surface of the impact bars, with a sputter duration of 10 s. The accelerating voltage of

the SEM was set to 10 kV and the samples were examined at a magnification of 1000x, in the

centre of the cross-section at break.

130

3.3 Results and Discussion

3.3.1 Solubility Parameters

The solubility parameters relating to PHBV, CA and TEC were developed from calculating the

cohesive forces between molecules, as outlined in Table 3.2, and can be a predictor of the solubility

parameter (δ) as well as the theoretical degree of miscibility. The cohesive forces are broken down

into the polar, dispersive and hydrogen bonding forces, derived using the Hoftyzer-van Krevelen

and Hoy methods 33. Some variation is seen in the solubility parameter for PHBV relative to that

reported by Snowdon et al., which can be the result of the method used to approximate the effect

of individual components of each molecule 34. Both Hoy and Hansen methods produce similar

solubility values for each polymer, suggesting they are theoretically soluble. Forster et al. report

that |Δδ|<2.0 MPa1/2 indicates miscibility, and |Δδ|>10.0 MPa1/2 is immiscible 35, thus it can be

concluded that CA and TEC are miscible, and PHBV, TEC and CA are all partially miscible with

each other. Therefore, a blend of PHBV, TEC and CA may be miscible and processable in

extrusion followed by injection moulding but does require experimental evaluation to confirm the

degree of miscibility.

Table 3.2. Solubility Parameters (δ) of CA, PHBV and TEC.

Sample Hoy Hoftyzer-Van Krevelen Hansen Average

δp δd δh δ δp δd δh δ δ δ

CA 15.6 14.3 13.0 24.8 0.5 20.9 14.4 25.6 25.1 36 25.1

PHBV 13.5 13.9 9.6 21.6 0.2 18.3 7.7 19.9 20.6 27 20.7

TEC 12.8 12.7 15.3 23.6 7.2 17.9 12.7 23.1 23.8 27 23.5

3.3.2 Thermal Properties

3.3.2.1 DSC

Plasticized CA has completely amorphous characteristics, having no melting peaks in the heating

part of the cycle and no crystallization peaks in the cooling part of the cycle. Literature reports that

the pCA melt peak thermogram appears like a Tg and does not conform to a traditional melting

peak, making enthalpy measurements unfeasable 25. However, no melting peak for pCA was

observed in this study. Thus, the crystallinity and melting peaks are only attributed to the presence

131

of PHBV. The Tg of PHBV was also not observed for any blend ratio by DSC, the result of the

heating scan rate being too rapid.

The DSC thermograms of PHBV/CA blends [Figure 3.1 (A)-(D)] illustrate the effect of cellulose

acetate on the PHBV melting and cooling cycles. In all cases, the Tg of PHBV and pCA were not

observed. The melting peak of PHBV and its enthalpy reduces with the addition of pCA, probably

due the absence of a melting peak structure in pCA. pCA appears to promote double melting peaks

in PHBV, although alternative factors can be responsible for the thermogram patterns; PHBV

measured with a low DSC heating rate is known to result in double melting peaks 37.

The formation of double melting peaks is attributed to the formation of a primary and secondary

crystals structure that results in the secondary crystals structure melting first 38. At 30 % pCA

loading, the melting peak is seen to broaden, indicating non-uniform crystal morphology.

Furthermore, the enthalpy is reduced due to absence of a melting peak related to CA. As the pCA

loading increases, the melting peak broadens and eventually diverges into a complete double

melting peak. Furthermore, the PHBV melting peak is decreased with greater pCA and TEC

content. Contrary to literature reports, TEC does not eliminate the double melting peak of PHBV

39, but significantly impacts the melting behaviour of PHBV, indicating it is not entirely associated

with cellulose acetate, and suggesting the double melting peak is the result of PHBV being non-

uniformly plasticized. TEC migrates to PHBV, which is also supported by the reduction in the

crystallization temperature and the enthalpy of crystallization during the cooling cycle, as indicated

in Table 3.3 and Figure 3.1 (C). The reduction in crystallinity seen in the PHBV/pCA blends can

be attributed to catastrophic degradation of PHBV by thermal hydrolysis due to the high processing

temperature. Molecular weight reduction has been reported to reduce the crystallinity after

extensive hydrolysis of PHBV 40.

132

-40 -20 0 20 40 60 80 100 120 140 160 180 200 220 240

PHBV/pCA (0/100)

PHBV/pCA (30/70)

PHBV/pCA (100/0)

PHBV/pCA (70/30)

En

do

Temperature (oC)

PHBV/pCA (50/50)

Double Melt Peak

(A)

-20 0 20 40 60 80 100 120 140 160 180 200 220 240

En

do

PHBV/pCA/CE (70/30/0.3)

PHBV/pCA (70/30)

PHBV/pCA/CE (50/50/0.3)

PHBV/pCA/CE (50/50)

(B)

PHBV/pCA/CE (30/70/0.3)

PHBV/pCA (30/70)

Temperature (oC)

-40 -20 0 20 40 60 80 100 120 140 160 180 200

PHBV/pCA (0/100)

PHBV/pCA (30/70)

PHBV/pCA (100/0)

PHBV/pCA (70/30)

En

do

Temperature (oC)

PHBV/pCA (50/50)

(C)

-40 -20 0 20 40 60 80 100 120 140 160 180 200

En

do

PHBV/pCA/CE (70/30/0.3)

PHBV/pCA (70/30)

PHBV/pCA/CE (50/50/0.3)

PHBV/pCA/CE (50/50)

(D)

PHBV/pCA/CE (30/70/0.3)

PHBV/pCA (30/70)

Temperature (oC)

Figure 3.1. DSC second heating curves of (A) PHBV/pCA blend ratios, and (B) PHBV/pCA/CE blends with 0.3

phr CE and DSC of cooling curve of (C) PHBV/pCA blend ratios, and (D) PHBV/pCA/CE blends with 0.3 phr

CE.

The addition of a chain extender to PHBV/pCA blends had no effect on the double melting peak

morphology, indicating secondary crystallite is still forming due to the presence of pCA [Figure

3.1 (B)]. The crystallinity of only PHBV is indicated in Table 3.3 and was slightly reduced in all

blend ratios, which indicates reduced chain mobility, reflecting the effect of the CE on PHBV 31.

With increased crosslinking and more complex molecular structures, the molecular weight

increased and the chain mobility reduced, inhibiting crystallization 41. Furthermore, the enthalp ies

of 50/50 and 30/70 polymer blends were taken over both melting peaks in DSC, illustrating the

133

fact that the overall enthalpy reduced significantly with the addition of pCA, which does not

crystallize. However, the crystallinity for the 30/70 PHBV/pCA blend and the 50/50 blend ratio

do not reflect the weight fraction of PHBV, indicating a secondary factor is reducing the ability of

PHBV to crystallize. Both blends were produced at the same temperature, however, PHBV is

known to be thermally sensitive, indicating chain scission of PHBV may have occurred. CE is not

able to significantly mitigate the issue efficiently (as shown in Figure 3.1 (B)).

Table 3.3. DSC results of PHBV/PCA blends with and without 0.3 phr CE.

PHBV/pCA Tc (°C) ∆Hc (J/g) Tm1 (°C) ∆Hm (J/g) Xc (%)

100/0 125.65 ±

0.52

94.13 ±

0.87

171.85 ± 1.26 89.55 ± 4.49 82.16

70/30 111.79 ± 0.30

60.38 ± 1.29

161.82 ± 0.01 64.50 ± 1.56 84.53

70/30/0.3 112.10 ±

1.08

57.81 ±

2.52

161.67 ± 1.36 63.73 ± 4.50 83.53

50/50 97.11 ± 0.09

40.16 ± 0.40

162.84 ± 0.11 41.52 ± 0.86 76.18

50/50/0.3 101.71 ±

1.32

37.51 ±

1.55

159.70 ± 0.28 39.55 ± 0.40 72.57

30/70 82.98 ± 0.13

17.24 ± 1.78

154.80 ± 0.54 20.86 ± 0.27 63.79

30/70/0.3 87.31 ± 0.72

19.88 ± 1.15

156.66 ± 0.44 19.80 ± 0.17 60.55

0/100 - 0 - 0 0

3.3.2.2 TGA

The TGA illustrates the thermal stability of the virgin polymers (PHBV and pCA) and their blends

in Figure 3.2 (A). Figure 3.2 (A) and (B) indicate a peak degradation at 285 °C and an onset of

degradation at 95 % mass of 267 °C for pristine PHBV, which is in agreement with literature 42.

pCA degradation has two peaks, one associated with the plasticizer and the other corresponding to

the polymer, correlating to approximately 303 and 368 °C, respectively.

TEC plasticizer has a broad degradation rate that slowly accelerates to a peak of approximate ly

241 °C (data not shown), in agreement with literature 43. Considering the broad range of TEC

degradation, the initial two peaks in the PHBV/pCA blend in Figure 3.2 (B) can be explained by

134

the degradation of unassociated and associated plasticizer, and are furthermore within the two

ranges reported by Ferfera-harrar and Dairi. These two ranges correspond to temperatures below

240 °C and between 240 to 300 °C when plasticizing cellulose acetate with TEC 44. The

degradation of the associated TEC is clearly illustrated by the 0/100 blend ratio at approximate ly

300 °C.

With the addition of pCA, the blend’s thermal stability is seen to improve and is compositiona lly

dependent, indicated by the curve shifting to higher temperatures. The resulting residue at 500 °C

is entirely from CA. However, pCA degradation onset is significantly earlier than virgin PHBV,

beginning at approximately 100 °C. The initial stages of thermal degradation are entirely the result

of TEC and any associated moisture, being the most sensitive component of the polymer blend as

illustrated in the TGA of CA, TEC and pCA in Figure 3.3.

The thermal degradation peaks of PHBV have been observed to shift towards higher temperatures

as the PHBV composition is reduced, which is attributed to the overlap of the PHBV degradation

curve and the degradation curve of the associated plasticizer in CA. However, the (50/50) blend

ratio does not follow the compositional trend. It must be noted that the (50/50) PHBV/pCA blend

was processed at 210-220 °C, significantly different from the (70/30) blend at 200-210 °C.

Degradation of PHBV molecular weight during processing into smaller and more volatile units is

exacerbated by the higher processing temperature and can result in early onset of degradation in

TGA, which is visible due to the higher percentage of PHBV relative to pCA, but is not found in

the PHBV/pCA (30/70) blend.

SA-GMA is a chain extender reported to improve the properties of PHBV when processed at

higher temperatures by increasing the activation energy and, therefore, improving the thermal

stability, as shown in Figure 3.2 (C) and (D) 31. The effect is most prominently displayed by the

increased pCA thermal degradation peaks for all PHBV/pCA blend ratios. An interesting factor is

displayed by the TEC degradation peak not overlapping with the PHBV degradation peak in the

PHBV/pCA/CE (70/30/0.3) blend, causing the PHBV degradation peak thermogram to follow that

of virgin PHBV. This suggests that TEC is almost entirely associated with pCA and degrades

separately from PHBV. Furthermore, the limits of a CE’s ability to rebuild the molecular weight

135

of PHBV was observed in the PHBV/pCA/CE (70/30/0.3) blend ratio. The degradation curve of

PHBV in the PHBV/pCA/CE (70/30/0.3) blend was decreased back to a temperature similar to

PHBV and degrading separately from the associated TEC in pCA.

50 100 150 200 250 300 350 400 450 500 550 600

0

20

40

60

80

100

120

Temperature (°C)

Wei

gh

t (%

)

50/50

70/30

30/70

0/100

(A)

100/0

100 150 200 250 300 350 400 450 500

0

1

2

3

4

5

6100/0

50/50

30/70

70/30

0/100

Der

iv. W

eigh

t (%

/°C

)Temperature (°C)

(B)

200 300 400

0

20

40

60

80

100

Wei

gh

t (%

)

Temperature (°C)

100/0

70/30

50/50

30/70

0/100

70/30/0.3

50/50/0.3

30/70/0.3

(C)

200 250 300 350 400

0

1

2

3

4

5

6

7 100/0

70/30

50/50

30/70

0/100

70/30/0.3

50/50/0.3

30/70/0.3

(D)

Der

iv.

Wei

gh

t (%

/°C

)

Temperature (°C)

Figure 3.2. TGA (A) and DTGA (B) of PHBV/pCA blends and TGA (C) and DTGA (D) of PHBV/pCA/CE

blends with 0.3 phr CE.

136

100 150 200 250 300 350 400 450 500

0

20

40

60

80

100

120

Temperature (°C)

Wei

gh

t (%

)

CA

pCA

(A)

TEC

100 150 200 250 300 350 400 450 500

0

1

2

3

4

pCA

TECCA

Der

iv. W

eigh

t (%

/°C

)

Temperature (°C)

(B)

Figure 3.3: TGA (A) and DTGA (B) of TEC, pCA and CA.

3.3.3 DMA

Figure 3.4 (A) and (B) illustrate the storage modulus of various PHBV/pCA blends and the

addition of Joncryl to blends, respectively. Naturally, PHBV is expected to have the highest storage

modulus, being a brittle thermoplastic polymer relative to pCA. The addition of a more flexib le

polymer such as plasticized cellulose acetate, which has one of the lowest storage moduli,

significantly reduced the storage modulus of PHBV in all blend ratios assessed. However, in 50/50

blend ratios, produced at a temperature of 210-220 °C, the storage modulus is slightly higher than

70/30 and 30/70 blend ratios which suggests an optimized point where the negative effect of pCA

content and partially plasticized PHBV on the blend stiffness is reduced.

The addition of a chain extender was intended to rebuild the molecular weight of PHBV due to the

high processing temperature, but no effect was seen for all blend ratios except 50/50, as shown in

Figure 3.4 (B). The effect of the chain extender on the storage modulus effectively reduced the

stiffness of PHBV/CA (50/50) blends to that of other blend ratios. A chain extender is known to

decrease crystallinity by inducing crosslinking, therefore, the increased molecular weight can

result in a less “glassy” blend with moduli like pCA 41. These results do not coincide with what is

expected of the mechanical properties as the overall blend mechanical ductility and flexibility have

not been improved relative to the neat constituents.

137

The tan(δ) was studied to determine the compatibility and potential interactions of a PHBV/CA

blend and was evaluated based on the peak position. Figure 3.4 (C) illustrates the tan(δ) of the

virgin PHBV, pCA and their three blends. Most notable is the fact that individual Tg’s are present,

indicating the blends are not miscible. The addition of 0.3 phr CE had no effect on the tan(δ), and

individual the Tg’s remained unaffected (Figure 3.4 (D)).

0 50 100 1500

1

2

3

4

5

Temperature (°C)

Sto

rag

e M

od

ulu

s (G

Pa

) 50/50

70/3030/70

0/100

(A)

100/0

0 50 100 1500

1

2

3

Sto

rag

e M

od

ulu

s (G

Pa

)

Temperature (°C)

50/50

100/0

70/30

70/300.3

0/100

50/50/0.3

30/70

30/70/0.3

(B)

0 50 100 150

0.0

0.2

0.4

0.6

0.8

1.0

-20 0 20 400.0

0.1(C)

Tan

()

Temperature (°C)

100/070/30

50/50

30/70

0/100

Tan

()

Temperature (°C)

100/0

70/30

50/50

30/70

0/100

0 50 100 1500.0

0.2

0.4

0.6

0.8

1.0

-20 0 20 400.0

0.1

100/0 70/30/0.3

70/30 50/50/0.3

50/50 30/70/0.3

30/70

0/100

Tan

()

Temperature (°C)

(D)

Tan

()

Temperature (°C)

Figure 3.4. Storage modulus of (A) PHBV/pCA blends and (B) PHBV/pCA/CE blends with 0.3 phr CE, and

Tan(δ) of (C) PHBV/pCA blend ratios and (D) PHBV/pCA/CE blends with 0.3 phr CE.

The Tg of PHBV measured from the peak in the temperature vs. tan(δ) curve, and reported in Table

3.4, is in agreement with literature reports 45, and the Tg of plasticized cellulose acetate is 116.5

138

°C. Literature reports the Tg of pCA from DSC analysis to be 98 °C 44. The difference is attributed

to the different methods of acquiring the Tg, where DMA is a dynamic thermo-mechanical measure

while DSC is a thermal measurement. The pCA Tg in PHBV/pCA 50/50 with and without CE was

not observable due to the drive force of DMA approaching 0 N and stopping the experiment.

The Tg of PHBV shifts towards lower temperature as the pCA ratio increases and, therefore, the

associated TEC in CA increases, as shown in Table 4. This indicates TEC migration from pCA to

PHBV, further confirming the findings from DSC. These indications also illustrate that the blends

are not partially miscible.

Table 3.4. Glass transition temperatures of PHBV/pCA blends with and without CE measured by DMA

analysis.

PHBV/pCA/CE Tg1 (°C) Tg2 (°C)

100/0 23.98 ± 0.19 -

70/30 -0.89 ± 0.19 -

70/30/0.3 -0.17 ± 1.94 -

50/50 -10.89 ± 0.74 -

50/50/0.3 -7.64 ± 3.22 -

30/70 -12.01 ± 2.97 132.32 ± 0.31

30/70/0.3 -14.41 ± 0.42 133.72 ± 0.38

0/100 - 116.51 ± 0.66

3.3.4 Mechanical Properties

3.3.4.1 Tensile and Flexural Properties

The tensile properties of virgin PHBV illustrated in Figure 3.5 to Figure 3.7 are in agreement with

literature reports; tensile strength of 30-40 MPa, tensile modulus of 2.3-3 GPa, elongation at break

of 3 % 42,46, with some variation in ductility and impact due to the specific processing conditions.

The results do, however, fit within the ranges of the materials data sheet provided by Eastman.

Park et al. reported a tensile stress at break of 70.0 MPa for cellulose acetate and triethyl citrate

blend with a ratio of 75/25, which is in good agreement with the experimentally found parameter.

The values of tensile modulus, elongation at yield, flexural strength and flexural modulus were

139

2200 MPa, 8.8 %, 65.4 MPa and 2370 MPa respectively, as reported by Park et al., which were

slightly lower than found in this study 28.

The introduction of pCA into PHBV as a blend, resulted in all mechanical properties achieving

neither of the individual constituents’ properties. The tensile properties of all blend ratios reduced

below those of PHBV and pCA. Buchanan et al. reported similar findings for blends of

compression moulded PHBV and cellulose acetate butyrate (CAB), where a ratio of PHBV/CAB

(50/50) resulted in lower tensile modulus and strength relative to either constituent. However, the

same ratio significantly improved the elongation at break in a narrow ratio percentage range, but

was not observed in this study 20.

The addition of 0.3 phr CE did not improve the tensile strength, however, as the CA content

increases, the tensile modulus is seen to marginally improve, approaching and exceeding pCA in

(50/50) and (30/70) blends. CE was introduced into PHBV prior to processing, indicating the effect

would largely be in PHBV. Thus, as the PHBV molecular weight is rebuilt, the tensile modulus

approaches that of virgin PHBV.

The flexural modulus and strength are the ability of the polymer to distribute the applied force

along the surface of the test sample which is subject to the maximum stress. The flexural modulus

and strength indicated in Figure 3.6 are negatively impacted in all blend ratios, although increased

pCA content does correlate with increased flexural strength in the blends. The chain extender is

seen to marginally improve the flexural modulus and strength of PHBV/pCA 50/50 and 30/70

blends. The improvement is negligible and may be due to the increased processing temperature

relative to PHBV/pCA 70/30 blends.

140

A B C D E F G H0

1

2

3

4

5

Tensile Modulus

Tensile Strength

PHBV/Plasticized CA

Ten

sile

Mo

du

lus

(GP

a)

0

10

20

30

40

50

60

70

80

90

Ten

sile

Str

en

gth

(M

Pa

)

Figure 3.5. Tensile modulus and strength of PHBV/pCA blends: (A) 100/0, (B) 70/30, (C) 50/50, (D) 30/70, (E)

0/100, (F) 70/30 + 0.3phr CE, (G) 50/50 + 0.3phr CE, and (H) 30/70 + 0.3phr CE.

A B C D E F G H0

1

2

3

4

5

6

7

8

Flexural Modulus

Flexural Strength

PHBV/pCA

Fle

xu

ral

Mo

du

lus

(GP

a)

0

10

20

30

40

50

60

70

80

90

Fle

xu

ral

Str

eng

th (

MP

a)

Figure 3.6. Flexural modulus and strength of PHBV/pCA blends: (A) 100/0, (B) 70/30, (C) 50/50, (D) 30/70, (E)

0/100, (F) 70/30 + 0.3phr CE, (G) 50/50 + 0.3phr CE, and (H) 30/70 + 0.3phr CE.

3.3.4.2 Notched IZOD Impact and Elongation at Break

Figure 3.7 illustrates the notched IZOD impact strength and elongation at break of PHBV, pCA

and PHBV/pCA blends, with and without 0.3 phr CE. The PHBV impact strength and elongation

at break are considerably lower than literature reports 42, but can be attributed to the difference in

the processing temperature profiles, as PHBV is known to be thermally sensitive. The pCA

141

elongation at break was found to be 16 %, 100 % greater than reported in literature, which is

attributed to the longer resting period for plasticized CA before processing. Pre-processing, Park

et al. rested the plasticized CA for 75 mins 28. The notched IZOD impact strength of pCA is below

that reported by Mohanty et al., due to the higher processing temperature used in this study 25.

Furthermore, the elongation at break of amorphous polymers does not always result in a high

notched IZOD impact strength 42. The elongation at break of PHBV/pCA blends does not show a

significant difference compared to virgin PHBV, which is a result of the poor chain mobility during

tensile testing. Furthermore, the impact strength does improve at higher pCA loadings, particular ly

with a 110 % improvement for PHBV/pCA (30/70) blend, which will be further investigated by

SEM morphology to check the interaction between PHBV and pCA. The chain extender did not

improve the impact properties of PHBV/pCA blends but mitigated the measured variation seen in

tested samples of the 30/70 blend.

A B C D E F G H0

5

10

15

20 Elongation at Break

Impact Strength

Elo

ngati

on

at

Break

(%

)

PHBV/pCA

0

10

20

30

40

Im

pact

Str

en

gth

(J/m

)

Figure 3.7. Elongation at break and notched IZOD impact strength of PHBV/pCA blends: (A) 100/0, (B) 70/30,

(C) 50/50, (D) 30/70, (E) 0/100, (F) 70/30 + 0.3phr CE, (G) 50/50 + 0.3phr CE, and (H) 30/70 + 0.3phr CE.

3.3.5 HDT and Density

The heat deflection temperature is the temperature where a polymer deflects 250 µm with 0.455

MPa applied force, and is directly correlated with the degree of crystallinity and the Tg. High

crystallinity of blends correlates to an HDT approaching the melt temperature. Table 3.5 indicates

HDT of 143 °C for PHBV to be in agreement with literature 34,42.

142

As plasticization of CA increases, the HDT is reported to drop, reaching 64 °C at a 30 % loading

47. In this study, the HDT of pCA with a 25 % TEC loading was found to be 81.1 ± 2.4 °C,

considerably lower than the Tg found through DMA. The TEC exists as a liquid and probably has

a Tg well below 0 °C, causing the discrepancy.

The HDT appears to be composition dependent, shifting towards lower temperatures with pCA

addition, parallel to the reducing crystallinity which is found for PHBV blends in literature 42. The

CE has no effect on the HDT of PHBV/pCA.

The density was observed to reduce in PHBV/pCA blends, which was attributed to PHBV and

TEC molecular scission resulting in increased porosity. Increased porosity can introduce

microcellular voids which may impede crack propagation, explaining the mechanical phenomenon

discovered before. However, CE addition increases the density, seemingly mitigating the porosity

in PHBV/pCA blends. CEs are known to rebuild the molecular weight and appears to have done

so for PHBV, however, density reduction is observed for (70/30) and (50/50) blend ratios,

warranting further investigation through SEM.

Table 3.5. Heat deflection temperature and density of PHBV/pCA/CE blends.

PHBV/pCA/CE HDT (°C) Density (c/cm3)

100/0 143.6 ± 2.0 1.236 ± 0.009

70/30 126.5 ± 1.9 1.179 ± 0.010

70/30/0.3 127.2 ± 1.7 1.179 ± 0.013

50/50 108.1 ± 6.8 1.182 ± 0.009

50/50/0.3 102.1 ± 6.0 1.182 ± 0.006

30/70 90.6 ± 0.2 1.248 ± 0.006

30/70/0.3 94.2 ± 0.9 1.256 ± 0.004

0/100 81.1 ± 2.4 1.288 ± 0.001

3.3.6 SEM

The structure of PHBV found in Figure 3.8 (A) has a similar morphology to that reported by Slongo

et al., the entire structure being homogenous with few if any imperfections on the impact surface.

The structure does show some stress cracks from impact testing, probably attributed to the higher

voltage and increased resolution of the micrograph 39. Figure 3.8 (E) illustrates pCA having a

143

delaminated structure and pullout from impact testing, the entire structure is also homogenous,

which is not reported in literature. As pCA loading increases, see Figure 3.8 (B) to (D), a crystalline

rigid structure forms, attributed to pCA, which also has sections of a delaminated structure.

However, there is no pattern of pullout. A significant amount of porosity is evident as well as non-

uniformity throughout the blends. Furthermore, Figure 3.8 (B) has evident phase separation,

supporting the DMA findings of complete immiscibility. Figure 3.8 (D) has no uniformity in

structure and porosity, which correlates with the variation observed in the impact strength.

The addition of CE to the blends, as shown in Figure 3.8 (F)-(H), clearly has a significant impact

on the morphology homogeneity. PHBV/pCA/CE (70/30/0) blend has reduced visible porosity,

although phase separation is still evident.

The SEM of PHBV/pCA (50/50) blend with 0.3 phr CE still appears to have the white crystal

structures randomly distributed on the surface of the impact fracture, but the sample has fewer

dark voids, and it is also difficult to discern an interface between PHBV and pCA. There is no

comparable morphology in literature for PHBV and CA blends of this kind. Visibly there is

reduced porosity in (50/50) and (30/70) blends with CE, however, since the density remains

unchanged, the pore sizes have reduced. Furthermore, SEM indicates the samples have become

morphologically homogeneous, suggesting some crosslinking has occurred between the polymers.

144

Figure 3.8. SEM morphology of PHBV/pCA/CE blend ratios: (A) 100/0, (B) 70/30, (C) 50/50, (D) 30/70, (E)

0/100, (F) 70/30 + 0.3phr CE, (G) 50/50 + 0.3phr CE, and (H) 30/70 + 0.3phr CE after break.

145

3.4 Conclusions

Biodegradable polymers, i.e. PHBV and cellulose acetate, were blended together in various

proportions, with and without chain extender, using a twin-screw extruder, and specimens were

prepared by injection moulding. The observed properties such as DSC, DMA and SEM

morphology showed that PHBV and pCA blends are completely immiscible at all blend ratios,

despite the calculated solubility parameters and literature review indicating potential miscibility.

The mechanical properties of all pCA blends were significantly reduced, however, the impact

strength exceeded that of virgin PHBV and pCA in blend ratios containing high pCA content. This

is attributed to PHBV degradation from the high processing temperature, which is reflected by the

increased porosity observed by SEM, reduced density, and a reduced measure of crystallinity

through DSC. Furthermore, the plasticizer TEC migrated during processing, partially plasticizing

PHBV, reducing its melt temperature and forming secondary crystallite structures. The unique Tgs

for DMA, SEM and DSC indicate that PHBV and CA are not miscible. PHBV/pCA 30/70 blend

ratios had a 110 % improvement in impact strength, resulting from partial foaming of the sample

and, consequently, leading to deterioration of the other mechanical properties relative to virgin

PHBV. Hence, PHBV/pCA blends need more research to achieve miscibility and to improve the

mechanical and thermal properties.

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(41) Slongo, M. D.; Brandolt, S. D. F.; Daitx, T. S.; Mauler, R. S.; Giovanela, M.; Crespo, J. S.;

Carli, L. N. Comparison of the Effect of Plasticizers on PHBV—and Organoclay—Based

Biodegradable Polymer Nanocomposites. J. Polym. Environ. 2018, 26 (6), 2290–2299.

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Kinetics, Crystallinity, and Surface Morphology. Mol. Cryst. Liq. Cryst. 2012, 556, 288–

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Crystallization, Melting Behavior and Morphology of Poly(Trimethylene Terephalate).

Polym. Test. 2007, 26 (2), 144–153. https://doi.org/10.1016/j.polymertesting.2006.08.011.

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151

Chapter 4: Marine Degradation Behaviour of PHAs and Natural

Fibres-based Biocomposites

Abstract

Worldwide, plastics are being disposed improperly each year, instigating environmental initiat ives

to combat the accumulation of plastics in the environment. Mismanaged plastic waste is shifted by

the elements, and a majority ends up in the world’s oceans. Accumulating plastics in oceans is

steadily increasing, resulting in consumer demands for environmentally sustainable and marine

biodegradable plastics. The research into marine biodegradable plastics is still in its infancy under

comparable standards but has garnered significant interest in the recent decade. Poly(3 -

hydroxybutyrate-co-3-hydrovalerate) (PHBV) and its biocomposites with Miscanthus (Misc)

fibres were developed and studied to ascertain if natural fibres can improve the marine

biodegradability and mechanical properties of polyhydroxyalkanoates. Injection moulded

PHBV/Misc 85/15 and 75/25 composites had improved the tensile and flexural moduli by 55 and

100% respectively due to Misc reinforcement. The notched Izod impact strength increased by

100% as compared to that of PHBV due to impeded crack propagation. Misc fibres minimally

affected the PHBV elemental composition and organic content as a result of elemental analysis.

Under ASTM standards D7991-15, the biodegradation of virgin PHBV, PHBV/Misc 85/15 and

PHBV/Misc 75/25 was simulated under a marine environment for the first time, and observed

biodegrading by 86%, 95% and 100%, respectively in 412 days. This study concludes that PHAs

are marine biodegradable and PHA-based biocomposites with natural fibres improves the marine

biodegradability of PHAs.

152

4.1 Introduction

In the evolving economy, bio-based plastics are making their mark and considered part of the new

economy among recycled commodity plastics1. The shift in economic objectives is driven by the

projected cumulative growth of primary plastic waste produced which will exceed 25 billion metric

tons in 20502. It has resulted in an expected 25 % growth in the production of bio-based and

biodegradable plastics is projected by 20233. For example, Bio-polyethylene terephthalate has

shown a growth from 7% to 80% of the global bioplastic production capacity between 2010 and

20174. For a majority of major plastic packaging producers, a goal of 100% recycled, compostable

or reusable plastic is set to be used in their products by 20255. However, biodegradable plastics

holds significant importance in combatting mismanaged plastic waste which is expected to become

double by 2025 6.

Biodegradation can be defined as a polymer biologically degrading by microorganisms (fungi or

bacteria) into carbon dioxide or methane, water and biomass7 by composting, soil biodegradation,

marine biodegradation or a combination of one of these processes and chemical or manmade

intervention. For example, a major mode of degradation for polyhydroxyalkanoates (PHAs) are

through enzymatic hydrolysis8. Such biodegradable polymers hold a significant advantage in

reducing the future potential plastic waste leakage and retention into the environment, with the

intention of letting the plastics degrade. It is already well documented that 86% of packaging waste

in 2015 was disposed of or littered, and 32% did not end up in a landfill or be incinerated9. These

unsustainable global approaches have resulted in significant increase to greenhouse gases in the

environment.

It has been found that renewable plastics have a significantly lower energy requirement during

production and a significantly lower carbon emission at end of life10. Therefore, bio-based

biodegradable polymers have the potential to combat climate change with sustainable production

methods and reduced impact after disposal. The most common bio-based biodegradable polymers

utilized in industry are cellulose acetate, poly(lactic acid) (PLA), thermoplastic starch based blends

and PHAs11.

153

Of the three main methods of biodegradation, composting is a synthetic mode where cellulose

acetate 12 and PLA degrade 13, and outside of the composting conditions, they show little if any

degradative behaviour. Composting has conditions at approximately 55 °C, compared to the

natural environment which may occasionally exceed 30 °C. Therefore, to truly minimize the plastic

waste environmental impact, PHAs and thermoplastic starches are ideal.

Aside from landfills, a reservoir of waste plastics ends up in the ocean, with a mild estimate of

15% waste plastic ending up in the ocean, by 2020 the ocean plastic will increase by 6.2 million

metric tons per year 6. This future forecast is what has been driving the recent research into marine

biodegradable polymers, as illustrated by Figure 4.1.

1995 2000 2005 2010 2015 2020

0

5

10

15

Nu

mb

er o

f P

ub

lica

tion

s

Year

Figure 4.1: Articles published in the area of polymer science, on the topic of "marine degradation of polymer"

from Web of Science (excluding review articles) (Obtained February 11, 2020).

PHAs are the most well-known marine biodegradable polymer 14, that is suited to reduce the long

term impact of ocean plastic waste. PHAs are degraded by a number of bacteria and fungi 15.

154

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a copolymer based of the

homopolymer poly(hydroxybutyrate), with hydroxyvalerate units which increase the amorphous

fraction and improve the susceptibility to enzymatic attack 8. Which is further confirmed when the

depolymerase activity improved with up to 40 % hydroxyvalerate content 16. However, to mainta in

the biodegradability of PHBV, other components must also be biodegradable. For example, non-

biodegradable polymers minimize the interactions between PHBV and enzymes, especially when

PHBV is the minor phase 17.

Compared to other biodegradable polymers (i.e. poly(ε-caprolactone) (PCL), poly(ethylene

adipate) (PEA), poly(ethylene succinate) (PES) etc.), PHBV can be degraded, in 28 days in both

fresh and saltwater provided the environmental conditions are suitable. Other PHAs such as PHB

and poly(3-hydrobutyrate-co-4-hydroxybutyrate) (PH4B) take slightly longer 18. PHBV also

shows biodegradable behaviour in soil 19, water or sand and a combination, which suggests a wide

distribution of PHA degraders in the environment 20. Thus, it is well established that PHAs, and

by virtue PHBV are biodegradable. The expected degradation mechanism of PHBV is illustrated

in Figure 4.2, where the final products are acetyl and propionyl attached to a synthesis co-enzyme

(CoA) to produce other molecules.

155

Figure 4.2: Expected pathway of PHBV degradation 20.

Typically PHBV is expensive to produce, in large part due to its poor availability in industry 11.

Therefore, price reduction is significant for utility in commodity applications. A method to

maintain the sustainability of bio-based polymers and reduce costs is to incorporate fibres or fillers

from the agricultural industry. It is well known that cellulose is marine biodegradable, as it is used

as a reference material, therefore, cellulose containing materials can be suitable as fillers in PHAs.

Under ASTM D6691-17, PHBV/CaCO3 and 20% seagrass fibre composites have shown improved

biodegradation rates due to poor matrix- fibre interfacial adhesion which improved physical

156

degradation and enhanced biological attack 21. Similarly starch incorporation into PHBV has

shown similar biodegradation rate improvements in natural marine waters over PHBV alone 22.

However, the major limitation of these studies is either ending the study before biodegradation

completion, modifications to the standard, or not applying a standard. Further studies on PHBV

composite marine biodegradation have not been found at this time, however, studies on soil

biodegradation can be used to supplement a hypothesis. PHBV and distillers dried grains with

solubles has shown to have 6 times the weight loss after 168 days in soil studies following no

ASTM standard23. PHBV and peach palm biocomposites similarly showed an accelerated rate of

biodegradation during soil burial tests following ASTM G160-9824. These studies reflect that

natural filler introduction into biodegradable polymers may improve the biodegradation of PHBV-

based biocomposites.

The new marine biodegradation standard, ASTM D7991-15, involves testing polymer samples in

lab using natural marine waters (unlike ASTM D6691-17). This method allows for testing of

samples under conditions reflecting either floating plastics, or plastics submersed in sediment in

coastal waters. Additional control parameters are light exposure and sample form (ground or solid).

As per our literature survey, marine biodegradation of PHAs following ASTM D7991-15 has not

been completed, and PHBV-based biocomposite marine biodegradation has not been determined

to completion with agricultural fibres and fillers. Therefore, the objectives of this research are to

study of marine biodegradability of PHBV-based biocomposites following ASTM D7991-15, and

succinctly determine if natural fibre reinforced PHBV has improved marine biodegradability.


4.2.1 Materials

PHBV pellets (ENMAT Y1000P), with 1-5% hydroxyvalerate), were purchased from Tianan

Biological Materials Co. Ltd. Miscanthus (Misc) grass fibres (New Energy Farms, Ontario,

Canada), had an average length and diameter of 4.65 ± 2.5 and 0.74 ± 0.02 mm respectively2 5 .

Marine water and sediment from the rainbow reef exhibit were kindly provided by Ripley’s

Aquarium in Toronto, Canada and used within 3 days of acquisition. For marine biodegradation

157

studies, BaOH2 was purchased from Fisher Scientific, HCl was purchased from LabChem and

BBOT was purchased from Thermo Scientific.

4.2.2 Composite Processing

Prior to processing Misc fibre and PHBV pellets were dried overnight at 80 °C. Composite

processing was completed in DSM Explore (DSM research, Netherlands), co-rotating twin screw

with a processing temperature of 180 °C, a fill pack and hold of 10 MPa, and a 2-minute retention

time at 100 rpm.

4.2.3 Elemental Analysis (CHNS)

Elemental analysis (C, H, N and S) of the cellulose, PHBV, PHBV/Misc and marine sediment was

completed using a Perkin-Elmer elemental analyser Flash 2000 (Thermo Scientific, USA). The

column was heated to 900 °C with carrier gases of helium and oxygen with flow rates of 140

mL/min and 250 mL/min respectively. Samples between 2-3 mg were taken and 4 replicates were

completed for each and 6 BBOT samples were measured to ascertain the calibration curve.


4.2.4.1 Differential Scanning Calorimetry (DSC)

PHVB and composites were analysed by DSC (Q200, TA instruments, Delaware, USA). Sample

weight of 5-10 mg were subjected to a heating rate of 10 °C/min from -40 to 200 °C. The Cooling

cycle was 10 °C/min to -40 °C, with isothermal conditions lasting 3 minutes. The melting

temperature (Tm) and enthalpy of melting (ΔHm) were observed from the first and second heating

cycles, respectively.

The % crystallinity (𝑋𝐶) of the first and second heating cycle of PHBV in polymer composites was

calculated using Equation 4.1 26.

158



) × 100 %

Equation 4.1

Where ∆𝐻𝑚0 is the theoretical enthalpy of 100 % crystalline PHBV (109 J/g) 27 and 𝑤𝑓 is the weight

fraction of PHBV.

4.2.4.2 Thermogravimetric Analysis (TGA)

TGA analysis (Q500, TA instruments, Delaware, USA), was completed on 15-20 mg samples of

virgin PHBV, Misc and the PHBV/Misc biocomposites. A heating rate of 10 °C/min until 700 °C

was applied under a N2 atmosphere. A purge and balance flow rate of 40 and 60 ml/min was

utilized. The ash content of the marine sediment and PHBV/Misc composites, were analysed under

air atmosphere at 900 °C, as per ASTM D2584.



Tensile and flexural samples were conditioned as per ASTM D618 for 48 hours. Characterizat ion

of mechanical properties was completed with an Instron 3382 (Massachusetts, USA). Tensile

samples were analysed with a testing rate of 5 mm/min, as per ASTM D638. Flexural samples

were tested following ASTM D790 with a span width of 52 mm.

4.2.5.2 Notched IZOD Impact Properties

Notched IZOD impact test samples were analysed by a Zwick Roell HIT25P (Ulm, Germany)

following the conditioning of samples as per ASTM D618. Each sample was tested following

ASTM D256 and the average of six replicates was taken as the impact strength.


Impact fractured samples were analysed by SEM utilizing a Phenom ProX desktop (Eindhoven,

Netherlands). All samples were gold sputter coated for 10 seconds by a Cressington Sputter Coater

159

(Watford, England), and analysed with a 10 kV accelerating voltage. Samples were analysed under

1000x magnification.

4.2.7 Contact Angle

The contact angle of PHBV and composites were determined using a ramé-hart goniometer 260-

U1. Utilizing the sessile drop technique, deionized water or diiodomethane were dropped upon the

surface of the injection moulded samples, functioning as a polar and non-polar testing liquid.

DROPimage software was utilized to measure the contact angles of three replicates for each sample

to determine the hydrophilicity of the neat polymer and composites.

4.2.8 Marine Biodegradation

Marine biodegradation was completed as per ASTM D7991-15. Marine water and sediment was

kindly donated by Ripley’s Aquarium in Toronto, from the rainbow fish exhibit. A ratio of 150 g

of marine water to 250 g of sediment was utilized and 100 mg of cryoground cellulose and test

samples were submersed in the sediment. Cotton cellulose filter paper without binders (Fisher

Scientific) was used as standard material for the validation criteria. The environment chamber was

maintained at 25 ± 2 °C. To ensure an airtight seal on the desiccators, petroleum jelly and parafilm

were used at the periphery of the lid after each titration. The initial conditions of the marine water

over an entire month before acquisition are outlined in Table 4.1.

Table 4.1: Marine water initial parameters

Temp

(°C) pH

Salinity

(µg/L)

NH3-N

(mg/L)

NO2-N

(mg/L)

Dissolved

Oxygen

(mg/L)

Alkalinity

(mg/L

CaCO3)

Nitrate

(mg/L)

23.55 ± 0.45

8.06 ± 0.03

31.86 ± 0.67

0.01 ± 0.01

0.004 ± 0.002

7.52 ± 0.07 164 ± 5 35 ± 7

160



Virgin PHBV had comparable mechanical properties (Figure 4.3) to literature, however, the

sample stiffness and strength is higher by approximately 25%, and the impact and elongation are

lower compared to literature, which are attributed to the processing conditions 28. PHBV/Misc

composites benefitted from increased tensile and flexural moduli by 55 and 100% (Figure 4.3 A)

and B)) which can be attributed to the high modulus of a single internode of Misc grass (12.1 GPa)

29. The tensile and flexural moduli reinforcement of Misc fibre of PHBV is well studied in literature

and known to minimally impact the tensile and flexural strength 30.

Furthermore, it was found that 15% and 25% Misc fibre increased the impact strength of PHBV

by 102% and 113%, however, these attributes are the result of PHBV requiring toss correction

(Figure 4.3 C)). The value of PHBV samples without toss correction was 21.77 J/m, still below

that of PHBV/Misc biocomposites. Natural fibre reinforcement has been found to increase the

impact strength of PHBV and some other polymers, however, it is dependent on the fibre

orientation, composition, type, and interfacial adhesion between the matrix and fibre 31–33. The

Misc fibre impedes crack propagation, which resulted in the improved impact strength 34.

Furthermore the long Misc fibres are stronger than PHBV, requiring them to absorb energy as they

are pulled out of the PHBV matrix.

161

100/0 85/15 75/250

2

4

6

8

10

12

PHBV/Miscanthus

Tensile Modulus

Tensile Strength

Ten

sile

Mo

du

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(GP

a)

A)

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40

60

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sile

Str

en

gth

(M

Pa

)100/0 85/15 75/25

0

2

4

6

8

10B)

PHBV/Miscanthus

Flexural Modulus

Flexural Strength

Fle

xu

ral

Mo

du

lus

(GP

a)

0

20

40

60

80

100

Fle

xu

ral

Str

eng

th (

MP

a)

100/0 85/15 75/250

2

PHBV/Miscanthus

C)

Elongation at Break

Notched Izod Impact

Elo

ng

ati

on

at

Brea

k (

%)

0

10

20

30

40

No

tch

ed

Izo

d I

mp

act

(J/m

)

Figure 4.3: PHBV/Misc A) tensile modulus and strength, B) flexural modulus and strength, and C) elongation

at break and impact strength.

4.3.2 DSC

The melt enthalpy and crystallinity of the second heating cycle of PHBV samples (Crystallinity of

Misc composites remains unchanged or slightly increased but no significant nucleating effect was

found, reflecting similar results in literature 37. Natural filler addition was reported to reduce the

crystallinity of the PHA weight fraction in biocomposites, by introducing chain disorder and

misalignment such that the crystalline region is unable to properly form around the Misc fibres

38,39. However, the aspect ratio of Misc fibre is larger, indicating a reduced nucleating effect.

162

The reduced crystallinity of the first heating cycle is a product of the processing conditions, which

indicates the amorphous PHBV fraction, microorganisms will be exposed too. Amorphous PHAs

have been reported to improve the biodegradation rate by enhancing the enzymatic and hydrolyt ic

degradation due to improved adsorption of enzymes and increased water penetration respectively

8,40–42. Since there is no change in crystallinity, any changes in biodegradation are the result of the

natural fibres or filler composition, morphology, interfacial adhesion between the matrix and filler,

and the matrix morphology.

Table 4.2) are within agreement of literature28,35. Deroiné et al. reported crystallinity of PHBV

from first heating cycle is slightly lower than the second, correlating with the thermal properties

of PHBV found 36. Crystallinity of Misc composites remains unchanged or slightly increased but

no significant nucleating effect was found, reflecting similar results in literature 37. Natural filler

addition was reported to reduce the crystallinity of the PHA weight fraction in biocomposites, by

introducing chain disorder and misalignment such that the crystalline region is unable to properly

form around the Misc fibres 38,39. However, the aspect ratio of Misc fibre is larger, indicating a

reduced nucleating effect.

The reduced crystallinity of the first heating cycle is a product of the processing conditions, which

indicates the amorphous PHBV fraction, microorganisms will be exposed too. Amorphous PHAs

have been reported to improve the biodegradation rate by enhancing the enzymatic and hydrolyt ic

degradation due to improved adsorption of enzymes and increased water penetration respectively

8,40–42. Since there is no change in crystallinity, any changes in biodegradation are the result of the

natural fibres or filler composition, morphology, interfacial adhesion between the matrix and filler,

and the matrix morphology.

Table 4.2: DSC of PHBV/Misc composites.

First Heating Cycle Second Heating Cycle

PHBV/

Misc Tm1 (°C) ∆Hm1 (J/g) Xc1 (%) Tm2 (°C) ∆Hm2 (J/g) Xc2 (%)

100/0 176.19 ± 3.99 75.06 ± 1.69 68.86 173.84 ± 0.83 78.94 ± 0.66 72.42

85/15 174.66 ± 1.51 63.61 ± 2.85 68.65 168.76 ± 0.10 68.81 ± 0.30 74.27

75/25 171.13 ± 0.38 57.74 ± 5.32 70.62 168.26 ± 0.91 62.55 ± 4.10 76.51

163

4.3.3 TGA

The peak degradation temperature of PHBV (Table 4.3), is within agreement of literature with

some variability 28,43. Misc fibre has a broad degradation peak (200-500 °C), associated with its

cellulose and hemicellulose. Thus, the thermal stability of PHBV/Misc composites is reduced,

however, it is the product of hemicellulose sensitivity to heat treatment 44 that initiates before

PHBV degradation.

The ash content of all biodegradation samples is within 1-2%, except cellulose which consists of

entirely organic content (Table 4.3). The sediment contains 59% inorganic content, while the

organic content is composed of the carbon, hydrogen, nitrogen and oxygen elements. From the ash

content, it is hypothesized that 100% of the reference sample, PHBV, and PHBV/Misc composites

will degrade to approximately 100%.

Table 4.3: TGA of marine biodegradation samples, Miscanthus and cellulose.

Sample Peak Degradation Temp. (°C) Ash Content (%)

Cellulose - 0.00

PHBV 302.99 1.59 ± 0.08

PHBV/Misc 85/15 292.85 354.42 1.43 ± 0.13

PHBV/Misc 75/25 284.28 355.95 1.81 ± 0.29

Miscanthus Fibre 358.16 1.49 ± 1.03

Sediment N/A 59.15 ± 0.30

4.3.4 Elemental Analysis

The cellulose elemental constituents’ (Table 4.4), carbon and hydrogen, are within agreement of

literature with a hydrogen/carbon molar ratio of 1.73 45. The residual oxygen is approximated as

49.39%, assuming the cellulose is entirely pure. Based on PHBV having approximately 2%

hydroxy valerate, the theoretical carbon, hydrogen and oxygen mass is 55.9, 7.0 and 37.1%

respectively, within agreement of the elemental analysis results (Table 4.4). The carbon mass (%)

of Misc fibres is approximated as 47-49% 46, and the difference in hydrogen content (6%) of

cellulose, hemicellulose and lignin is negligible 45. The PHBV/Misc composite elemental analysis

is not expected to differ significantly from PHBV. The sediment contains approximately 11%

164

carbon, while the residual compounds make up hydrogen and the potential oxygen. From the TGA

analysis, it is approximated at 39.55% oxygen.

Table 4.4: Elemental analysis of marine biodegradation samples and sediment.

Sample Carbon (%) Hydrogen (%) Nitrogen (%) Sulphur (%) H/Ca

Cellulose Filter Paper 43.94 ± 1.17 6.40 ± 0.16 0.05 ± 0.00 0.00 ± 0.00 1.73

PHBV 56.93 ± 1.77 7.05 ± 0.10 0.46 ± 0.06 0.00 ± 0.00 1.48

PHBV/Misc 85/15 55.04 ± 0.28 6.94 ± 0.04 0.55 ± 0.01 0.00 ± 0.00 1.50

PHBV/Misc 75/25 55.55 ±0.64 6.79 ± 0.08 0.54 ± 0.04 0.00 ± 0.00 1.46

Sediment 11.20 ± 0.13 0.10 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.11

a Molar Ratio.

4.3.5 SEM Morphology

SEM morphology of impact fractured PHBV sample (Figure 4.4 A)) reflects a smooth,

undamaged, homogeneous surface. Similar results were also found for virgin PHBV samples in

literature 47. Misc fibres populate the PHBV/Misc biocomposites, distributing layered fibres

throughout the structure (Figure 4.4 B) and C)). Some evidence of Misc fibre pull-out, which may

have resulted in the increase in impact properties. The long fibres have sustained their structural

integrity, and with pull-out, channels can be made throughout the polymer matrix, exposing more

enzyme activity sites on the PHBV surface. Furthermore, in case fibres break (Figure 4.4 2B)),

they provide exfoliated carbon sources, which enhance the fibre degradation processes.

165

Figure 4.4: Notched IZOD Cross-Section SEM Morphology of A) PHBV, B) PHBV/Misc 85/15 and C)

PHBV/Misc 75/25 impact samples after break.

4.3.6 Contact Angle

The contact angle of cellulose using both solutions was zero due to the high porosity, because the

original form is filter paper as per ASTM D7991-15 standard (Table 4.5). The contact angle of

PHBV using deionized water or diiodomethane is within agreement of the reported literature 48.

With the introduction of Misc fibre, it appears the hydrophobicity and hydrophilicity increased,

however, the surface roughness increased, impacting the sessile drop measures. Therefore, the

measurement of contact angle for PHBV/Misc composites was not accurately feasible. Wu et al.

has documented PHA biocomposites and in all cases the water absorption rate increased with

increasing load of natural fibres 49.

Table 4.5: Contact angles of polymer composites and cellulose filter paper.

Contact Angle (deg)

Polymer/Composite Deionized Water Diiodomethane

Cellulose 00.0 ± 0.0 00.0 ± 0.0

PHBV 66.7 ± 1.8 39.1 ± 2.6

PHBV/Misc 85/15 73.1 ± 0.4 42.8 ± 1.6

PHBV/Misc 75/25 73.0 ± 1.8 44.4 ± 0.6

166


Marine biodegradation of cellulose and PHBV/Misc biocomposites was completed and supports

the hypothesis that natural fibre inclusion into PHBV matrix will improve the biodegradation rate.

Figure 4.5 A) indicates PHBV and PHBV/Misc biocomposites have a greater CO2 evolution

compared to cellulose due to their 12% higher carbon content. Furthermore, the initial degradation

rate of cellulose and PHBV/Misc biocomposites were metabolically similar due to the quantity of

CO2 produced, which was attributed to the presence of cellulose and hemicellulose in Misc. The

CO2 evolution of PHBV has a consistent rate throughout biodegradation that was slower than both

composites and cellulose.

The biodegradation of the cellulose samples under ASTM D6691 were reported to take a longer

period 50, however, this can be attributed to the fact that soil/sand provides a greater microbiome

that can accelerate biodegradation than water alone 20. PHBV biodegradation under ASTM D6691

has been completed, but at a temperature of 30 °C 41, such that the temperature enhanced the

polymer enzymatic interactions and the overall biodegradation 51. PHBV biodegradation at 25 °C,

PHBV/Misc 85/15 and 75/25 biocomposites have 15 and 25% faster biodegradation compared to

virgin PHBV (Table 4.6). PHBV/Misc 75/25 is also 100% marine biodegradable in 412 days,

under ASTM D7991 standard. This level of improvement is significantly lower compared to

reported literature in other forms of biodegradation medium, where a 100% improvement can be

seen with 10-20% natural fibre loadings 52. However, such studies were carried out with whole

fibres, where the morphology of the fibre is significantly important, in addition to the different

environment.

167

0 50 100 150 200 250 300 350 400 450 500

0

20

40

60

80

100

120

140

160

180

200

220

Cellulose

PHBV

PHBV(15% Misc)

PHBV(25% Misc)

CO

2 E

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on

(m

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Time (day)

A)

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30

40

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70

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110B)

% B

iod

eg

ra

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tio

n

Time (day)

Cellulose

PHBV

PHBV(15% Misc)

PHBV(25% Misc)

Validation Criteria for Experiment (60%)

Figure 4.5: Cellulose and PHBV/Misc biocomposites A) CO2 evolution and B) overall biodegradation.

Table 4.6: Marine biodegradation results.

Sample CO2 Evolved

(mg)

Theoretical CO2

(mg)

Biodegradation

(%) Time (day)

Cellulose 152.15 161.45 96.38 450

PHBV 180.44 208.96 89.42 450

PHBV/Misc 85/15 192.27 201.83 97.11 450

PHBV/Misc 75/25 204.73 203.67 100.52 412

4.4 Conclusion

Introducing 15 and 25% Misc natural fibres into PHBV increases the tensile and flexural moduli

by 55% and 100%, while also increasing the impact properties by 100% without affecting the

tensile and flexural strength. PHBV and Misc fibres complement each other by having similar

hydrogen to carbon ratios and ash content, such that total CO2 evolved will remain constant with

fibre addition.

Biodegradation is a complex process depending on the environment and the polymer or composite

properties. The thermal properties of PHBV remained relatively unchanged, indicating the effects

of the fibres in biodegradation are related to their composition and morphology, and that there is

no expected improvement in the amorphous fraction of PHBV by Misc to enhance biodegradation

processes. However, after breakage of the samples, exfoliated Misc fibre is exposed, in addition

to channels from Misc fibre pull-out, increasing the surface area for biodegradation processes.

168

PHBV tested to ASTM D7991-15 in a mixture of marine water and sediment biodegrades by 86%

in 412 days. The addition of 15 and 25% Misc fibres into PHBV increases the biodegradation of

PHBV by approximately 15 and 24%, respectively in 412 days. Furthermore, PHBV/Misc 75/25

is 100% marine biodegradaton in 412 days, as per ASTM D7991. Therefore, to implement

sustainable plastic technologies in industry, enhancing PHBV with Misc fibres can improve the

mechanical and biodegradable properties which can increase the utility of biodegradable plastics

and reduce the future build-up of plastics in oceans.

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Chapter 5: Marine Degradation Behaviour of PHAs and Distillers

Dried Grains with Solubles Biocomposites

Abstract

Plastic waste worldwide is being improperly disposed of, leading to great environmental concerns

about its accumulation in the environment. Plastic accumulates in the ocean, making human

intervention difficult, which has lead to an increased consumer demand in marine biodegradable

plastics. Poly(3-hydroxybutyrate-co-3-hydrovalerate) (PHBV) and PHBV-based biocomposites

with DDGS were developed and studied to determine if the presence of protein can improve the

biodegradation of PHBV biocomposites. DDGS was washed of water-soluble compounds and

injection moulded with PHBV in ratios of PHBV/DDGS 85/15 and 75/25. Tensile/flexural strength

of PHBV/DDGS 85/15 and 75/25 reduced by 30 and 40% respectively compared to PHBV. Tensile

and flexural moduli pf PHBV/DDGS biocomposites remained unchanged relative to PHBV.

DDGS increased the nitrogen content, and therefore the predicted protein content in PHBV/DDGS

biocomposites, which is predicted to improve the microbial growth. Morphological and thermal

properties are indicated to not improve the biodegradation processes due to unchanged crystallinity

and minor improvement in surface area. The marine biodegradation of PHBV/DDGS 85/15 and

75/25 biocomposites was performed as per ASTM D7991-15, resulting in an improved

biodegradation rate of 15 and 40% compared to PHBV, respectively in 295 days. PHBV/DDGS

75/25 biodegradation exceeded cellulose within 150 days and is 100% marine biodegradable in

295 days. This study indicates PHBV/DDGS biodegradation is more effective than

PHBV/Miscanthus biodegradation at similar natural fibre/filler loadings.

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5.1 Introduction

As the world’s economy is redirected to more sustainable sources of plastics, the research and

development of new or unexplored plastics becomes paramount to replace petroleum-based

plastics. To approach zero emissions, and become sustainable, the plastics must be bio-based and

biodegradable. The growing desire for carbon neutrality has led to 25% projected growth in the

production of bio-based and biodegradable plastics by 2023 1. Usage of bio-based plastics not only

reduces the carbon emissions, but also the energy production requirements 2, however, it must be

coupled with biodegradability to combat mismanaged waste 3. Its projected by 2050, 99% of all

seabirds will have ingested plastic waste 4.

Biodegradation of polymers is a biotic process involving bioassimilation of biodegradation

products following enzymatic hydrolysis. Abiotic factors such as oxidation, hydrolyt ic

degradation, thermal degradation and mechanical degradation are all processes that further

enhance biotic degradation 5,6. What makes biodegradable polymers valuable and commercia l ly

viable is when they can degrade under natural environment conditions 7. The common bio-based

and biodegradable plastics used commercially are cellulose acetate, poly(lactic acid) (PLA),

thermoplastic starch and PHAs 8. However, both PLA and cellulose acetate are only compostable

9,10, leaving thermoplastic starch and PHAs suited for natural environments.

Polyhydroxyalkanoates (PHAs) make up a wide range of bio-based biodegradable polymers 11,

with varying properties dependent on the combination of monomers. PHAs are biodegradable in

both aerobic and anaerobic environments under a variety of conditions 12 and by both bacteria and

fungi 13, unlike PLA and other petroleum-based biodegradable polymers (i.e. PCL, PBS, PBAT

etc.). PHAs are particularly attractive to combat the growing plastic waste ending up in the ocean

each year 3. Due to their suitability for biodegradation in many environments, PHAs can form a

small cradle to grave cycle that can minimize the impact of plastic waste on the environment 14.

Two basic PHAs are poly(hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-

hydroxyvalerate) (PHBV) and are the most common used industrially. PHBV is an improvement

upon PHB by introducing a greater amorphous fraction which improves water absorption 15 and

175

enzymatic depolymerase activity 16,17. As the amorphous region in HV increases, so too does the

biodegradation 18–20. The marine biodegradation of PHBV can vary depending on the type of

environment, taking anywhere from 28 days to 100 days or more depending on the conditions 17,21.

However, the cost of PHA production is also significantly higher than conventional and bio-based-

biodegradable plastics 22, which is a detriment to the research on optimal applications in industry.

Blending PHAs with other biodegradable polymers can reduce costs, but is usually undesirable as

it may impact biodegradability or the sustainability. The incorporation of natural fibres has long

since been used to reduce cost and increase performance in automotive applications. Therefore,

PHA biocomposites can benefit from the research and potentially improve the biodegradability

and sustainability. Natural fibres and fillers are mainly composed of cellulo se, hemicellulose and

lignin, which have been reported to improve the biodegradation of PHAs in aerobic environments

23–25. However, protein-based fillers provide an additional attribute that increases the complexity

of biodegradation. Distillers dried grains with solubles (DDGS) contain 30% protein before any

treatment 26, which does contain beneficial forms of nitrogen that can be used by microorganisms

(Figure 5.1). During thermal processing, protein denaturation and degradation does occur,

releasing several compounds trapped in the polymer matrix and fibres, mainly consisting of CO2,

NH3, H2O and other sulphides 27. Ammonia readily forms equilibrium with ammonium during

biodegradation which can be used as a nitrogen source 28. Furthermore, ammonia can also be

absorbed and utilized by microorganisms in the nitrogen cycle as nitrates 28, after ammonia

oxidation and nitrite oxidation 29. Other forms of thermal degradation products and any residual

protein is depolymerized by microorganisms, trafficked through the cellular membrane 30 and used

to form amino acids 31.

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Figure 5.1: Proteinaceous Filler Bio-assimilation by Microorganisms 27.

The potential of PHBV/DDGS based biocomposites has not been explored in marine

biodegradation, however, PHA/soy and PHA/DDGS biocomposites have been found to have

improved or comparable soil biodegradation properties to those of PHA/starch biocomposites in

similar filler ratios 32. PHA/DDGS and PHA/soy have also proven successful in composting

studies, showing complete degradation in 84 days 24.

As per our literature survey, PHA/DDGS marine biodegradation studies have not been completed

in any form. Therefore, the objectives of this research are to study of marine biodegradability of

PHBV-based biocomposites containing DDGS following ASTM D7991-15, to determine the

extent protein based natural fillers can improve the biodegradation of PHBV.

177


5.2.1 Materials

PHBV ENMAT Y1000P pellets (1-5% hydroxyvalerate content), were purchased from Tianan

Biological Materials Co. Ltd. DDGS was sourced from CG Tech and washed with room

temperature tap water for 15 minutes following the methodology outlined by Zarrinbakhsh et al.

26. Marine sediment and marine water were kindly provided from the rainbow reef exhibit of

Ripley’s Aquarium in Toronto, Canada, and BaOH2 and HCl were purchased from Fisher

Scientific and LabChem respectively. For Marine biodegradation sample characterization, BBOT

was purchased from Thermo Scientific.

5.2.2 Composite Processing

Before processing, PHBV pellets and DDGS were dried overnight at 80 °C. PHBV/DDGS

composites were first compounded in a twin-screw extruder (Leistritz, Germany) with a barrel

temperature of 180 °C and a screw speed of 100 rpm. The pelletized extrudates were dried

overnight for 24 hours before injection moulding in an Arburg injection moulding machine

(ARBURG allrounder 370C, Germany). The barrel had a temperature profile of 45 to 180 °C.

5.2.3 Elemental Analysis (CHNS)

Marine sediment, cellulose filter paper, PHBV, and PHBV/DDGS composite elements (C, H, N

and S) were determined using a Perkin-Elmer elemental analyser Flash 2000 (Thermo Scientific,

USA). Argon and oxygen carrier gases with flow rates of 140 mL/min and 250 mL/min were used

respectively. Samples between 2-3 mg were taken and 4 replicates were completed for each at an

oven temperature of 950 °C. 6 BBOT samples were measured to determine the calibration curve.

178


5.2.4.1 Differential Scanning Calorimetry (DSC)

PHBV/DDGS biocomposites were analysed by DSC (Q200, TA instruments, USA). The heating,

cooling and heating cycle were completed at 10 °C/min, over the range of -40 to 200 °C. Sample

weights of 5-10 mg were used and the melting temperature (Tm) and enthalpy of melting (ΔHm)

were observed from the first and second heating cycles.

The PHBV and PHBV/DDGS composite % crystallinity (𝑋𝐶) from the first and second heating

cycle was calculated using Equation 5.1 33.



) × 100 %

Equation 5.1

Where 𝑤𝑓 is the weight fraction of PHBV and the theoretical enthalpy ∆𝐻𝑚0 of 100 % crystalline

PHBV (109 J/g) 34.

5.2.4.2 Thermogravimetric Analysis (TGA)

TGA analysis (Q500, TA instruments, USA), was completed with a purge and balance flow of 40

and 60 mL/min, under a nitrogen atmosphere, with sample masses of 15-20 mg. A heating rate of

10 °C/min was used until 700 °C. The ash content of the marine sediment for ASTM D7991-15

and the DDGS composites, were analysed as per ASTM D2584.



An Instron 3382 (Instron, USA) was used to characterize the tensile and flexural properties.

Tensile and flexural samples were analysed following ASTM D638 at 5mm/min and ASTM D790

with a span width of 52 mm respectively. All samples were conditions following ASTM D618.

179

5.2.5.2 Notched IZOD Impact Properties

Notched IZOD impacts samples were conditioned as per ASTM D618. Samples were tested as per

ASTM D256 using a Zwick Roell HIT25P (Zwick Roell, Germany) with six replicates. Toss

correction was performed on all samples below 27 J/m.


Impact samples after break were gold sputter coated for 10 seconds using a Cressington Sputter

Coater (Cressington, England). The impact break surface was analysed by SEM using a Phenom

ProX desktop (Phenom World, Netherlands) with an accelerating voltage of 10 kV. Samples were

analysed at 1000x magnification.

5.2.7 Contact Angle

The surface contact angle of samples was studied using the Sessile drop technique with

diiodomethane and deionized water. The contact angle was measured by a ramé-hart goniometer

260-U1 (ramé-hart instrument co., USA), and DROPimage software to determine the surface

hydrophobicity.


Following ASTM D7991-15, marine biodegradation of PHBV, PHBV/DDGS composites and

cellulose filter paper (Thermo Fischer) was completed. Cryoground test samples (100mg) were

submersed in 150 g marine water and 250 g marine sediment 3 months after acquisition of the

marine sediment and water from the rainbow reef exhibit at Ripley’s Aquarium, Toronto. The

marine biodegradation experiment was conducted at a temperature of 25 ± 2 °C, and each

desiccator was sealed with parafilm, and petroleum jelly. The initial conditions of the marine water

before acquisition are outlined in Table 4.1.

180

Table 5.1: Marine Water Parameters.

Temp

(°C)

pH Salinity

(µg/L)

NH3-N

(mg/L)

NO2-N

(mg/L)

Dissolved

Oxygen

(mg/L)

Alkalinity

(mg/L

CaCO3)

Nitrate

(mg/L)

23.55 ± 0.45

8.06 ± 0.03

31.86 ± 0.67

0.01 ± 0.01

0.004 ± 0.002

7.52 ± 0.07

164 ± 5 35 ± 7



Virgin PHBV had moduli and strength approximately 25% higher with a lower impact and

elongation at break compared to literature 35, which can be attributed to the processing conditions

(Figure 5.2 A) and B)). PHBV/DDGS 85/15 and 75/25 biocomposites tensile and flexural strength

reduced by 30 and 40% respectively, while the tensile and flexural moduli remained unchanged.

The reinforcement effect of DDGS in PHBV/PBS blends reports similar effects 36.

PHBV/DDGS 85/15 and 75/25 impact strength and elongation at break reduced by 25 and 55%

respectively (Figure 5.2 C)), showing an enhancement of crack propagation during impact studies.

DDGS is a multi- layered grain-based filler with a low aspect ratio compared to long fibres.

Furthermore, DDGS has incredibly poor mechanical properties with a strength modulus of 0.3-0.5

MPa and 2.41-5.24 MPa respectively 37, which provide less of an impedance on crack propagation

than virgin PHBV would.

181

100/0 85/15 75/250

1

2

3

4

5

PHBV/DDGS

A)

Tensile Modulus

Tensile Strength

Ten

sile

Mo

du

lus

(GP

a)

0

10

20

30

40

Ten

sile

Str

en

gth

(M

Pa

)

100/0 85/15 75/250

1

2

3

4

5

Flexural Modulus

Flexural Strength

Fle

xu

ral

Mo

du

lus

(GP

a)

B)

PHBV/DDGS

0

20

40

60

80

Fle

xu

ral

Str

eng

th (

MP

a)

100/0 85/15 75/250

1

2

3

4

5

Elongation at Break

Notched Izod Impact

Elo

ng

ati

on

at

Bre

ak

(%

)

0

10

20

30

PHBV/DDGS

C)

No

tch

ed I

zod

Im

pa

ct (

J/m

)

Figure 5.2: PHBV/DDGS A) Tensile Modulus and Strength, B) Flexural Modulus and Strength, and C)

Elongation at Break and Impact Strength.

5.3.2 DSC

The melt enthalpy and (%) crystallinity of neat PHBV samples from the second heating cycle

(Table 5.2) is within agreement of literature 35,38 and the first heating cycle enthalpy and

crystallinity is reported to be lower due to the processing 39. DDGS inclusion in the second heating

cycle acts like a nucleating agent, increasing the crystallinity which has been reported in

polypropylene composites 40, contrary to literature reporting PHA/DDGS biocomposites having

reduced PHBV crystallinity. However, the variation can be due to the high shear of the DSM which

can reduce the DDGS size compared to compression moulding techniques 41.

182

DDGS inclusion shows no additional reduction in the PHBV crystallinity of the first heating cycle,

indicating it is not optimal at inhibiting PHBV crystallization compared to larger fillers and fibres.

Greater amorphous PHBV fractions can potentially improve the water penetration and overall

biodegradation, however, the effect would be reduced due to DDGS nucleating effect.

Table 5.2: DSC of PHBV/DDGS Composites.

First Heating Cycle Second Heating Cycle

PHBV/

DDGS Tm1 (°C) ∆Hm1 (J/g) Xc1 (%) Tm2 (°C) ∆Hm2 (J/g) Xc2 (%)

100/0 177.00 ± 1.08 75.36 ± 1.73 69.14 173.85 ± 1.77 80.95 ± 0.54 74.26

85/15 174.07 ± 2.67 66.24 ± 0.93 71.49 169.79 ± 0.19 73.70 ± 1.97 79.54

75/25 172.23 ± 2.52 53.63 ± 1.05 71.72 168.98 ± 0.15 66.78 ± 2.21 81.68

5.3.3 TGA

The peak degradation temperature of virgin PHBV (Table 5.3), is similar to reports in literature

35,42. DDGS has two peaks associated to the hemicellulose and cellulose, consisting of

approximately 20 and 29% of the DDGS, similar to those reported in literature 26. The thermal

stability of DDGS has reduced slightly, however, it is due to the hemicellulose and protein

sensitivity to heat 27,43 that initiates before PHBV degradation.

The ash content of DDGS is similar to reports in literature 26, and by incorporating it into PHBV,

the inorganic content increases (Table 5.3). Cellulose is the only sample with no measurable ash

content by TGA analysis. The sediment contains 59% inorganic content, which can be attributed

to any non-carbonaceous minerals. Based on the ash content approximately 98-100% of the

samples must biodegrade for the PHBV and PHBV/DDGS samples to be considered 100%

biodegradable.

183

Table 5.3: TGA of Marine Biodegradation Samples, DDGS and Cellulose.

Sample Peak Degradation Temp. (°C) Ash Content (%)

Cellulose - 0.00

PHBV 302.99 1.59 ± 0.08

PHBV/DDGS 85/15 297.47 1.70 ± 0.25

PHBV/DDGS 75/25 287.08 350.97 1.94 ± 0.10

DDGS 288.73 348.94 3.25 ± 1.58

Sediment N/A 59.15 ± 0.30

5.3.4 Elemental Analysis

The cellulose sample elemental components (Table 5.4), carbon and hydrogen, are within

agreement of literature with a hydrogen/carbon molar ratio of 1.73 44. Nitrogen and sulphur are

negligible in cellulose and the residual oxygen is approximated as 49.39%. Based on PHBV having

approximately 2% hydroxy valerate, the theoretical carbon, hydrogen and oxygen mass is 55.9%,

7.0% and 37.1% respectively, within agreement of the elemental analysis results (Table 5.4). The

carbon, hydrogen, nitrogen, sulphur and oxygen of DDGS are reported as 43.83, 6.77, 5.37, 1.01

and 34.15% 45, which is expected to result in a reduced carbon content and increased nitrogen

content. Multiplying the nitrogen content by 6.25 using food analysis techniques 46, the

approximate protein composition of DDGS is 33.56%, which constitutes almost entirely of the

measured protein content 26. However, our results indicate a significantly higher nitrogen content,

which will significantly promote microbial growth through protein synthesis. The significant

presence of protein in PHBV/DDGS biocomposites implies a nitrogen source will be readily

available as either protein, thermally degraded protein or protein degradation products ammonia

27.

The sediment contains approximately 11% carbon and 0.11% hydrogen. Based on TGA analysis,

the remaining organic compounds make up approximately 39.55% oxygen.

184

Table 5.4: Elemental Analysis of Marine Biodegradation Samples and Sediment.

Sample Carbon (%) Hydrogen (%) Nitrogen (%) Sulphur (%) H/Ca

Cellulose 43.94 ± 1.17 6.40 ± 0.16 0.05 ± 0.00 0.00 ± 0.00 1.73

PHBV 56.93 ± 1.77 7.05 ± 0.10 0.46 ± 0.06 0.00 ± 0.00 1.48

PHBV/DDGS 85/15 54.83 ± 0.13 5.97 ± 0.71 6.66 ± 0.03 0.00 ± 0.00 1.30

PHBV/DDGS 75/25 54.16 ±0.09 5.25 ± 0.41 7.64 ± 0.76 0.00 ± 0.00 1.16

Sediment 11.20 ± 0.13 0.10 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.11

a Molar Ratio.

5.3.5 SEM Morphology (To be completed and SEM of samples)

SEM morphology of virgin PHBV (Figure 5.3 A)) impact samples have a smooth homogeneous

surface. DDGS inclusion into PHBV results in multiple small particles distributed throughout the

structure (Figure 5.3 B) and C)). Minor evidence of DDGS pull-out is observed, however, the

particles are small and thus not expected to improve the impact properties. Furthermore, the DDGS

particles have a rough surface and there is evidence of particle breakage occurring indicating the

poor mechanical strength of DDGS. These aspects may result in improved surface area which can

maximize the enzymatic activity.

Figure 5.3: Notched IZOD Cross-Section SEM Morphology of A) PHBV, B) PHBV/DDGS 85/15 and C)

PHBV/DDGS 75/25 Impact Samples After Break.

185

5.3.6 Contact Angle

Unground cellulose samples were in the form of filter paper, porous and thus both contact angle

measures were zero because diiodomethane and deionize3d water permeated the membrane (Table

5.5). The diiodomethane and deionized water contact angles are within agreement of literature,

and indicate slightly more hydrophobic surface characterstics which can be unfavourable for water

permeation 47. With the introduction of DDGS, there does appear to be a slight increase in surface

hydrophobicity, however, this characteristic is only present at high loadings of DDGS. This can

be attributed to the high protein component which may be hydrophobic.

There is potential the protein may reduce the water permeation at the surface of PHBV/DDGS

composites, reducing the potential rate of biodegradation. However, literature reports DDGS to

significantly improve the water absorption of PHBV 48. Therefore, water absorption and

penetration in PHBV/DDGS biocomposites is not only based on the surface hydrophilicity but

other characteristics such as the DDGS porosity.

Table 5.5: Contact Angles of Polymer Composites and Cellulose Filter Paper.

Contact Angle (deg)

Polymer/Composite Deionized Water Diiodomethane

Cellulose 00.0 ± 0.0 00.0 ± 0.0

PHBV 66.7 ± 1.8 39.1 ± 2.6

PHBV/DDGS 85/15 66.6 ± 1.2 39.7 ± 1.6

PHBV/DDGS 75/25 70.1 ± 0.9 37.3 ± 1.5


DDGS inclusion into PHBV is shown to improve the rate of biodegradation by a significant extent,

clearly exceeding PHBV and cellulose with 25% DDGS loadings. Figure 5.4 A) illustrates

increased CO2 evolution for PHBV and PHBV/DDGS composites compared to cellulose, due to

its high carbon content determined in elemental analysis. The initial rate of CO2 evolution for all

samples are similar, indicating a similar metabolic rate, however, after day 50, PHBV/DDGS 75/25

CO2 evolution exceeds cellulose. PHBV and PHBV/DDGS samples have approximately 20-25%

theoretical maximum CO2 evolution (Table 5.6), resulting in a greater quantity of CO2 to be

evolved.

186

Cellulose biodegradation is indicated to take a longer period of time in marine biodegradation

under ASTM D6691 49. Different inoculums can provide different diversity of microorganisms

with cellulolytic capabilities. Furthermore, soil/sand provides a greater microbiome that can

accelerate biodegradation relative to marine water alone 50.

PHBV biodegradation with 5-12% hydroxyvalerate at a temperature of 30 °C, under ASTM D6691

is reported to take 115 days to achieve 90-100% biodegradation 17. However, the faster rate can be

a characteristic of the hydroxyvalerate content and/or the temperature which enhances polymer -

enzyme interactions 51. As per our literature survey this is the first PHBV sample tested at 25 °C

as per a marine ASTM standard. In marine biodegradation studies the incorporation of 25% starch

into PHBV has been studied, and shows biphasic biodegradation and a 50% increase the rate over

32 days 52. DDGS contains approximately 5% starch, the remaining constituents being mainly

protein, cellulose, hemicellulose and lignin 26 which do not have high hydrophilicity like starch.

10% DDGS incorporation into PHA has bene studied in soil, resulting in a 5-6 times improvement

in initial mass loss and performing better than 10% starch loadings 32,53. PHA/DDGS 80/20 is also

reported to compost 47-70% faster than PHA/Lignin, PHA/Jute, PHA/Lyocell and PHA Hemp in

home composting conditions 24,54. However, all studies on PHA/DDGS biodegradation follow no

repeatable standards. PHBV/DDGS 85/15 and 75/25 biocomposites biodegrade approximately 15

and 40% faster than PHBV, respectively in 295 days, and PHBV/DDGS 75/25 exceeds the

biodegradation rate of cellulose after 150 days (Table 5.6). PHBV/DDGS 75/25 is 100%

biodegradable in 295 days under ASTM D7991-15.

187

0 50 100 150 200 250 300 350 400

0

20

40

60

80

100

120

140

160

180

200

220

CO

2 E

vo

luti

on

(m

g)

Time (day)

Cellulose

PHBV

PHBV(15% DDGS)

PHBV(25% DDGS)

A)

0 50 100 150 200 250 300 350 400

0

10

20

30

40

50

60

70

80

90

100

110B)

% B

iod

egra

da

tio

n

Time (day)

Cellulose

PHBV

PHBV(15% DDGS)

PHBV(25% DDGS)

Validation Criteria for Experiment (60%)

Figure 5.4: Cellulose and PHBV/DDGS Biocomposites A) CO2 Evolution and B) Overall Biodegradation.

Table 5.6: Marine Biodegradation Results.

Sample CO2 Evolved

(mg)

Theoretical CO2

(mg)

Biodegradation

(%) Time (day)

Cellulose 152.15 161.45 96.38 450

PHBV 180.44 208.96 89.42 450

PHBV/DDGS 85/15 175.76 201.44 90.37 361

PHBV/DDGS 75/25 206.72 196.59 101.41 295

5.4 Conclusion

Introducing 15% and 25% DDGS into PHBV results in an increased bio-based content, with

a reduction in the tensile/flexural strength by 30 and 40% respectively. The moduli remained

188

unchanged, indicating PHBV/DDGS biocomposites are suitable for rigid applications where

biodegradation is critical. DDGS also introduces an increased nitrogen content in the form of

proteins which may help in microbial growth during biodegradation.

The thermal characteristics of PHBV/DDGS biocomposites remained unchanged, except for

a DDGS nucleating effect in the second heating cycle. The crystallinity of the PHBV fraction in

PHBV and PHBV/DDGS biocomposites was 70%, indicating the amorphous PHBV fraction

microorganisms will encounter during biodegradation. There is no expected morphological or

thermal improvement by DDGS incorporation into PHBV which can enhance biodegradation

processes. However, DDGS physically is a multi- layered grain-based material, easing

fragmentation which may improve biodegradation rates.

PHBV under ASTM D7991, in a mixture of marine water and sediment biodegrades by 76% in

350 days. 15% and 25% DDGS loadings in PHBV improve the biodegradation rate of PHBV by

15 and 40%, respectively in 295 days. Higher loadings of DDGS significantly improve the

biodegradation rate, exceeding cellulose within 150 days. PHBV/DDGS biocomposites are

suitable for rigid plastic applications and reduce the future marine plastic waste build-up.

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Chapter 6: Overall Conclusions and Future Work

6.1 Overall Conclusions

This research was intended to explore the biodegradation properties of PHAs with the

addition of other bio-based fibres, fillers and polymers for commercial applications. The

sustainability of petroleum-based plastics has extensive detriments, from production to disposal

that effects the climate and the ecosystem. Using PHAs, the greenhouse gas emissions are

significantly lower, and they biodegrade in all types of environments. However, the costs of PHAs

are significantly greater than other bio-based polymers and petroleum-based polymers. Therefore,

the aim of this study was to incorporate cellulose based fibres, fillers and polymers into PHBV and

study the effect on the mechanical, thermal, morphological and biodegradable properties.

In the first study, cellulose acetate (CA) was blended with PHBV to study the miscibility,

mechanical, thermal and morphological properties. CA is a low-cost biopolymer, readily available

around the world, making it attractive to use with PHBV.

CA was plasticized with 25% triethyl citrate (TEC) based on literature to form pCA.

CA, TEC and PHBV are all partially miscible with each other based on solubility

calculations.

DMA indicated unique Tgs suggesting PHBV and pCA were not miscible.

Due to the high processing temperature, PHBV degraded which is reflected in a high

porosity observed in SEM.

TEC migrated during processing, partially plasticizing PHBV resulting in secondary

crystallite structures observed in DSC.

50 and 70% pCA improved the impact strength of PHBV by approximately 100% but

partial foaming of blends resulted in deterioration of the other mechanical properties.

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The second study focused on incorporating Miscanthus fibre into PHBV to study the effects

on marine biodegradation properties. It is established in literature that natural fibres can improve

the soil biodegradation and compost rate of PHBV. However, marine biodegradation studies

following a repeatable standard have not been found containing PHBV biocomposites with

Miscanthus fibre.

The first marine biodegradation study of PHBV following ASTM D7991-15 (as per our

literature survey) was completed and successfully proved that PHBV has marine

biodegradable behaviour in marine water and sediment.

15 and 25% Miscanthus natural fibres increased the biodegradation rate of

PHBV/Miscanthus by 15 and 24% respectively. PHBV/Miscanthus 75/25 is 100% marine

biodegradation in 412 days under ASTM D7991.

15 and 25% Miscanthus fibre increased PHBV tensile/flexural moduli by 55 and 100%

respectively. Impact properties of PHBV increased by 100% with 15 and 25% Miscanthus

fibre addition and tensile and flexural strength remained the same.

The third study investigated the effect of proteinaceous natural filler in PHBV and if they

can promote biodegradation of PHBV in marine environment to a greater extent than other natural

fibres. Distillers dried grains with solubles (DDGS) is a secondary agricultural residue made from

corn after ethanol production processes.

15 and 25% DDGS reduced the tensile/flexural strength by 30 and 40% respectively. The

modulus remained unchanged.

PHBV fraction crystallinity was not reduced with higher filler loadings, indicating DDGS

has a nucleating effect.

PHBV degraded by 86% in 195 days, while 15 and 25% DDGS loadings increased the

PHBV/DDGS marine biodegradation rate by 15 and 40%, respectively within that period.

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25% DDGS content improved PHBV/DDGS biocomposite biodegradation, exceeding

cellulose, and is 100% marine biodegradation in 295 days

The overall biodegradation results indicate that natural fibre and filler based of agri-residues

can improve the marine biodegradation of PHBV. Natural fibres can improve the impact and

modulus of PHBV at higher loadings. Natural fibres and fillers can reduce the cost of PHBV

significantly. More sustainable PHBV based biocomposites can be developed to combat climate

change and minimize the impact of plastics on the marine ecosystem.

Miscanthus fibre and DDGS can be used as a sustainable low-cost filler in biodegradable

polymers.

The use of DDGS and Miscanthus provides potential profit to farmers and industr ia l

ethanol manufacturing plants.

Miscanthus fibre can reinforce the impact and modulus of PHBV without affecting the

strength.

Miscanthus and DDGS both improve the marine biodegradation of PHBV. Furthermore, it

was determined that 25% DDGS improves the biodegradation of PHBV to a greater extent

than 25% Miscanthus fibre.

Development of PHBV based biocomposites can reduce the future magnitude of plastic

waste in the marine environment.

6.2 Future Work

The research completed in this study has identified the benefits on the marine

biodegradation, mechanical and thermal properties of natural fibre and fillers effects in PHBV

while also producing lower cost and more sustainable materials for commercial applications. This

research clearly indicates the benefits to the marine ecosystem and climate change, about the

implementation of sustainable bio-based and biodegradable biocomposites.

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The next steps to be investigated is the miscibility of PHBV and cellulose acetate.

A more stable plasticizer that will not migrate, may potentially resolve the issue of their

immiscibility based on the solubility calculations.

Potential biodegradable properties are required to determine if they’re marine

biodegradable or only in other environments.

Research into this area can indicate the potential for a simple low-cost method to reduce

PHBV while also giving some flexible properties.

PHBV biocomposites also require further research to examine their potential for commercia l

applications of single use plastics where their biodegradable properties would benefit the most.

PHBV biocomposites are suitable for rigid applications, however flexible packaging

applications have yet to be explored.

For commercial applications, large scale production methods must be determined to assess

if PHBV based biocomposites are viable.

A life cycle analysis can indicate the energy required to produce and dispose of

biodegradable polymers in a sustainable method to indicate the benefits of bio-based

polymers.

Additional biodegradation studies in different marine conditions or other natural

environments can indicate the benefits of PHBV biocomposites.

Documents

Biodegradable Poly(3-hydroxybutyrate-co-3-hydroxyvalerate