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 Materials and Methods ................................................................................................. 156
4.2.1 Materials................................................................................................................ 156
4.2.2 Composite Processing ........................................................................................... 157
4.2.3 Elemental Analysis (CHNS) ................................................................................. 157
4.2.4 Thermal Properties ................................................................................................ 157
4.2.5 Mechanical Properties........................................................................................... 158
4.2.6 Scanning Electron Microscopy (SEM) ................................................................. 158
4.2.7 Contact Angle ....................................................................................................... 159
viii
4.2.8 Marine Biodegradation ......................................................................................... 159
4.3 Results and Discussion................................................................................................. 160
4.3.1 Mechanical Properties........................................................................................... 160
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
4.3.7 Marine Biodegradation ......................................................................................... 166
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 Materials and Methods ................................................................................................. 177
5.2.1 Materials................................................................................................................ 177
5.2.2 Composite Processing ........................................................................................... 177
5.2.3 Elemental Analysis (CHNS) ................................................................................. 177
5.2.4 Thermal Properties ................................................................................................ 178
5.2.5 Mechanical Properties........................................................................................... 178
5.2.6 Scanning Electron Microscopy (SEM) ................................................................. 179
5.2.7 Contact Angle ....................................................................................................... 179
5.2.8 Marine Biodegradation ......................................................................................... 179
5.3 Results and Discussion................................................................................................. 180
5.3.1 Mechanical Properties........................................................................................... 180
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
5.3.7 Marine Biodegradation ......................................................................................... 185
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.
15 PHA Non-ASTM, Farmland Topsoil
25 °C, 35% Moisture (w.b.)
Plate 120 35% Mass Loss 216
16 PHA Non-ASTM, Farmland Topsoil
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
Film 110 80% Biodegradation 177
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
Film 110 90% Biodegradation 177
12 PHB ISO 14855-1, Mature
Compost
Film 45 80% Biodegradation 176
13 PHBV(3% HV) 81% Biodegradation
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,
Woods Hole Harbor Water
30 °C Film 100 80% Mineralization 163
8 PH4B(47% 4HB)
82% Mineralization
9 PHBV(8%
HV)
ASTM D6691,
Woods Hole Harbor Water
30 °C Film 100 85% Mineralization 163
10 PHBV(12% HV)
100% Mineralization
11 PHBV(8%
HV)
Non-ASTM, Lorient
Harbour
25 °C Film 180 36% Mass Loss 175
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
28 °C Film 86 71% Mass Loss 235
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 %)
Protocol and Source Form Days Results Ref.
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
Powder 12 67% Biodegradation 188
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
Powder 61 88% Biodegradation 181
8 141 95% Biodegradation
9 PHBV(8% HV) ASTM D5210-91,
Wastewater
Film 40 89% Biodegradation 240
10 PHB Non-ASTM, Domestic Sewage Sludge
Powder 21 90% Biodegradation 137
11 PHB Non-ASTM, Sewage
Sludge
Powder 16 87% Biodegradation 15
12 PHBV(13% HV) 96% Biodegradation
13 PHBV(20% HV) 83% Biodegradation
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.)
Film 60 (i) 65%, (ii) 82% Mass Loss
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.)
Film 140 (i) 74%, (ii) 89% Mass Loss
190
10 PHA-g-MA/Silane Treated Tea Plant Fibre (i) 80/20, (ii)
60/40
Non-ASTM, Farmland Topsoil
35% Moisture
(w.b.)
Film 140 (i) 67%, (ii) 81% Mass Loss
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
Non-ASTM, Farmland Topsoil
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.
PHA (co-monomer %)/Composite
Protocol and Source Form Days Results Ref.
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.
PHA (co-monomer %)/Composite
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.
2.8 References
(1) Geyer, R.; Jambeck, J. R.; Law, K. L. Production, Use, and Fate of All Plastics Ever Made.
Sci. Adv. 2017, 3 (7), 25–29. https://doi.org/10.1126/sciadv.1700782.
(2) Nova Institute. European Bioplastics https://www.european-bioplastics.org/ (accessed Mar
11, 2020).
(3) New Plastic Economy. The New Plastic Economy 2019 Progress Report; 2019.
(4) Storz, H.; Vorlop, K.-D. Bio-Based Plastics: Status, Challenges and Trends. Landbauforsch.
Volkenrode 2014, 63 (4), 321–332. https://doi.org/10.3220/LBF_2013_321-332.
(5) Masnadi, M. S.; El-Houjeiri, H. M.; Schunack, D.; Li, Y.; Englander, J. G.; Badahdah, A.;
Monfort, J. C.; Anderson, J. E.; Wallington, T. J.; Bergerson, J. A.; et al. Global Carbon
Intensity of Crude Oil Production. Science (80-. ). 2018, 361 (6405), 851–853.
https://doi.org/10.1126/science.aar6859.
(6) Gironi, F.; Piemonte, V. Bioplastics and Petroleum-Based Plastics: Strengths and
Weaknesses. Energy Sources, Part A Recover. Util. Environ. Eff. 2011, 33 (21), 1949–1959.
https://doi.org/10.1080/15567030903436830.
(7) Gallo, F.; Fossi, C.; Weber, R.; Santillo, D.; Sousa, J.; Ingram, I.; Nadal, A.; Romano, D.
Marine Litter Plastics and Microplastics and Their Toxic Chemicals Components : The Need
for Urgent Preventive Measures. Environ. Sci. Eur. 2018, 30 (1).
https://doi.org/10.1186/s12302-018-0139-z.
83
(8) Harding, K. G.; Dennis, J. S.; von Blottnitz, H.; Harrison, S. T. L. Environmental Analysis
of Plastic Production Processes: Comparing Petroleum-Based Polypropylene and
Polyethylene with Biologically-Based Poly-β-Hydroxybutyric Acid Using Life Cycle
Analysis. J. Biotechnol. 2007, 130 (1), 57–66.
https://doi.org/10.1016/j.jbiotec.2007.02.012.
(9) Deloitte Sustainability. Blueprint for Plastics Packaging Waste: Quality Sorting and
Recycling Final Report; 2018.
(10) Badia, J. D.; Gil-Castell, O.; Ribes-Greus, A. Long-Term Properties and End-of-Life of
Polymers from Renewable Resources. Polym. Degrad. Stab. 2017, 137, 35–57.
https://doi.org/10.1016/j.polymdegradstab.2017.01.002.
(11) ACS. Discovery Report The Future of Plastic; Drahl, C., Ed.; 2020.
(12) Chu, L. M. Landfills. In Encyclopedia of Ecology; Elsevier, 2008; pp 2099–2103.
https://doi.org/10.1016/B978-008045405-4.00345-1.
(13) Agamuthu, P. Landfilling in Developing Countries. Waste Manag. Res. 2013, 31 (1), 1–2.
https://doi.org/10.1177/0734242X12469169.
(14) Department of Health. Important Things to Know About Landfill Gas
https://www.health.ny.gov/environmental/outdoors/air/landfill_gas.htm (accessed Jan 13,
2020).
(15) Budwill, K.; Fedorak, P. M.; Page, W. J. Methanogenic Degradation of Poly(3-
Hydroxyalkanoates). Appl. Environ. Microbiol. 1992, 58 (4), 1398–1401.
(16) Rayne, S. The Need for Reducing Plastic Shopping Bag Use and Disposal in Africa. African
J. Environ. Sci. Technol. 2008, 3 (3), 1–3.
(17) Reviews of Environmental Contamination and Toxicology, Volume 227; Whitacre, D. M.,
Ed.; Reviews of Environmental Contamination and Toxicology; Springer Internationa l
Publishing: Cham, 2014; Vol. 227. https://doi.org/10.1007/978-3-319-01327-5.
84
(18) Tharpes, Y. L. International Environmental Law : Turning the Tide on Marine Pollution.
Univ. Miami Inter-American Law Rev. 1989.
(19) Cierjacks, A.; Behr, F.; Kowarik, I. Operational Performance Indicators for Litter
Management at Festivals in Semi-Natural Landscapes. Ecol. Indic. 2012, 13 (1), 328–337.
https://doi.org/10.1016/j.ecolind.2011.06.033.
(20) Wilcox, C.; Van Sebille, E.; Hardesty, B. D.; Estes, J. A. Threat of Plastic Pollution to
Seabirds Is Global, Pervasive, and Increasing. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (38),
11899–11904. https://doi.org/10.1073/pnas.1502108112.
(21) Cózar, A.; Echevarría, F.; González-Gordillo, J. I.; Irigoien, X.; Úbeda, B.; Hernández-
León, S.; Palma, Á. T.; Navarro, S.; García-de-Lomas, J.; Ruiz, A.; et al. Plastic Debris in
the Open Ocean. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (28), 10239–10244.
https://doi.org/10.1073/pnas.1314705111.
(22) Law, K. L.; Moret-Ferguson, S.; Maximenko, N. A.; Proskurowski, G.; Peacock, E. E.;
Hafner, J.; Reddy, C. M. Plastic Accumulation in the North Atlantic Subtropical Gyre.
Science (80-. ). 2010, 329 (5996), 1185–1188. https://doi.org/10.1126/science.1192321.
(23) Van Sebille, E.; England, M. H.; Froyland, G. Origin, Dynamics and Evolution of Ocean
Garbage Patches from Observed Surface Drifters. Environ. Res. Lett. 2012, 7 (4).
https://doi.org/10.1088/1748-9326/7/4/044040.
(24) Guedri-Knani, L.; Gardette, J. L.; Jacquet, M.; Rivaton, A. Photoprotection of
Poly(Ethylene-Naphthalate) by Zinc Oxide Coating. Surf. Coatings Technol. 2004, 180–
181, 71–75. https://doi.org/10.1016/j.surfcoat.2003.10.039.
(25) Jambeck, J.; Geyer, R.; Wilcox, C.; Siegler, T. R.; Perryman, M.; Andrady, A.; Narayan,
R.; Law, K. L. Plastic Waste Inputs from Land into the Ocean. 2015, 347 (6223).
https://doi.org/10.1126/science.1260352.
(26) EPA. Composting At Home https://www.epa.gov/recycle/composting-home (accessed Jan
85
13, 2020).
(27) World Centric. Compostable Plastics
http://www.worldcentric.org/biocompostables/bioplastics (accessed Apr 25, 2020).
(28) Environmental Protection Agency. An Analysis of Composting as an Environmental
Remediation Technology; 1998.
(29) Gibbens, S. What you need to know about plant-based plastics
https://www.nationalgeographic.com/environment/2018/11/are-bioplastics-made-from-
plants-better-for-environment-ocean-plastic/ (accessed Mar 11, 2020).
(30) Song, J. H.; Murphy, R. J.; Narayan, R.; Davies, G. B. H. Biodegradable and Compostable
Alternatives to Conventional Plastics. Philos. Trans. R. Soc. B Biol. Sci. 2009, 364 (1526),
2127–2139. https://doi.org/10.1098/rstb.2008.0289.
(31) Gutierrez-Wing, M. T.; Stevens, B. E.; Theegala, C. S.; Negulescu, I. I.; Rusch, K. A.
Aerobic Biodegradation of Polyhydroxybutyrate in Compost. Environ. Eng. Sci. 2011, 28
(7), 477–488. https://doi.org/10.1089/ees.2010.0208.
(32) Rudin, A.; Choi, P. Biopolymers. In The Elements of Polymer Science & Engineering;
Elsevier, 2013; pp 521–535. https://doi.org/10.1016/B978-0-12-382178-2.00013-4.
(33) Babu, R. P.; O’Connor, K.; Seeram, R. Current Progress on Bio-Based Polymers and Their
Future Trends. Prog. Biomater. 2013, 2 (1), 8. https://doi.org/10.1186/2194-0517-2-8.
(34) Voevodina, I.; Kržan, A. Bio-Based Polymers.
(35) Chen, G. Q. Plastics from Bacteria; 2010; Vol. 14. https://doi.org/DOI 10.1007/978-3-642-
03287_5_3.
(36) Kim, Y. J.; Choi, S. Y.; Kim, J.; Jin, K. S.; Lee, S. Y.; Kim, K. J. Structure and Function of
the N-Terminal Domain of Ralstonia Eutropha Polyhydroxyalkanoate Synthase, and the
Proposed Structure and Mechanisms of the Whole Enzyme. Biotechnol. J. 2017, 12 (1).
86
https://doi.org/10.1002/biot.201600649.
(37) Sungyeap Hong, C. L. An Overview of the Synthesis and Synthetic Mechanism of Poly
(Lactic Acid). Mod. Chem. Appl. 2014, 02 (04). https://doi.org/10.4172/2329-
6798.1000144.
(38) Greene, J. Biodegradation of Biodegradable and Compostable Plastics under Industria l
Compost, Marine and Anaerobic Digestion. Ecol. Pollut. Environ. Sci. 2018, 1 (1), 13–18.
(39) Braungart, M.; McDonough, W.; Bollinger, A. Cradle-to-Cradle Design: Creating Healthy
Emissions - a Strategy for Eco-Effective Product and System Design. J. Clean. Prod. 2007,
15 (13–14), 1337–1348. https://doi.org/10.1016/j.jclepro.2006.08.003.
(40) Kuciel, S.; Kuźniar, P.; Nykiel, M. Biodegradable Polymers in the General Waste Stream -
The Issue of Recycling with Polyethylene Packaging Materials. Polimery/Polymers 2018,
63 (1), 31–37. https://doi.org/10.14314/polimery.2018.1.5.
(41) Steinbüchel, A.; Valentin, H. E. Diversity of Bacterial Polyhydroxyalkanoic Acids. FEMS
Microbiol. Lett. 1995, 128 (3), 219–228. https://doi.org/10.1016/0378-1097(95)00125-O.
(42) Al-Salem, S. M.; Lettieri, P.; Baeyens, J. Recycling and Recovery Routes of Plastic Solid
Waste (PSW): A Review. Waste Manag. 2009, 29 (10), 2625–2643.
https://doi.org/10.1016/j.wasman.2009.06.004.
(43) Braunegg, G.; Lefebvre, G.; Genser, K. F. Polyhydroxyalkanoates, Biopolyesters from
Renewable Resources: Physiological and Engineering Aspects. J. Biotechnol. 1998, 65 (2–
3), 127–161. https://doi.org/10.1016/S0168-1656(98)00126-6.
(44) Visakh, P. M. Polyhydroxyalkanoates (PHAs), Their Blends, Composites and
Nanocomposites: State of the Art, New Challenges and Opportunities. RSC Green Chem.
2015, 2015-Janua (30), 1–17. https://doi.org/10.1039/9781782622314-00001.
(45) Choi, J.; Lee, S. Y. Efficient and Economical Recovery of Poly(3-Hydroxybutyrate) from
RecombinantEscherichia Coli by Simple Digestion with Chemicals. Biotechnol. Bioeng.
87
1999, 62 (5), 546–553. https://doi.org/10.1002/(SICI)1097-
0290(19990305)62:5<546::AID-BIT6>3.0.CO;2-0.
(46) Rebah, F. Ben; Prévost, D.; Tyagi, R. D.; Belbahri, L. Poly-β-Hydroxybutyrate Production
by Fast-Growing Rhizobia Cultivated in Sludge and in Industrial Wastewater. Appl.
Biochem. Biotechnol. 2009, 158 (1), 155–163. https://doi.org/10.1007/s12010-008-8358-1.
(47) Ramsay, B. A.; Saracovan, I.; Ramsay, J. A.; Marchessault, R. H. A Method for the Isolation
of Microorganisms Producing Extracellular Long-Side-Chain Poly (β-Hydroxyalkanoate )
Depolymerase. J. Environ. Polym. Degrad. 1994, 2 (1), 1–7.
https://doi.org/10.1007/BF02073481.
(48) Juengert, J.; Bresan, S.; Jendrossek, D. Determination of Polyhydroxybutyrate (PHB)
Content in Ralstonia Eutropha Using Gas Chromatography and Nile Red Staining. Bio-
Protocol 2018, 8 (5), 1–15. https://doi.org/10.21769/bioprotoc.2748.
(49) Koller, M.; Maršálek, L.; de Sousa Dias, M. M.; Braunegg, G. Producing Microbia l
Polyhydroxyalkanoate (PHA) Biopolyesters in a Sustainable Manner. N. Biotechnol. 2017,
37, 24–38. https://doi.org/10.1016/j.nbt.2016.05.001.
(50) Kourmentza, C.; Plácido, J.; Venetsaneas, N.; Burniol-Figols, A.; Varrone, C.; Gavala, H.
N.; Reis, M. A. M. Recent Advances and Challenges towards Sustainab le
Polyhydroxyalkanoate (PHA) Production. Bioengineering 2017, 4 (2), 1–43.
https://doi.org/10.3390/bioengineering4020055.
(51) Korotkova, N.; Lidstrom, M. E. Connection between Poly-β-Hydroxybutyrate Biosynthes is
and Growth on C1 and C2 Compounds in the Methylotroph Methylobacterium Extorquens
AM1. J. Bacteriol. 2001, 183 (3), 1038–1046. https://doi.org/10.1128/JB.183.3.1038-
1046.2001.
(52) Vroman, I.; Tighzert, L. Biodegradable Polymers. Materials (Basel). 2009, 2 (2), 307–344.
https://doi.org/10.3390/ma2020307.
88
(53) Chen, J.; Li, W.; Zhang, Z. Z.; Tan, T. W.; Li, Z. J. Metabolic Engineering of Escherichia
Coli for the Synthesis of Polyhydroxyalkanoates Using Acetate as a Main Carbon Source.
Microb. Cell Fact. 2018, 17 (1), 1–12. https://doi.org/10.1186/s12934-018-0949-0.
(54) Chen, Q.; Wang, Q.; Wei, G.; Liang, Q.; Qi, Q. Production in Escherichia Coli of Poly(3-
Hydroxybutyrate-Co-3-Hydroxyvalerate) with Differing Monomer Compositions from
Unrelated Carbon Sources. Appl. Environ. Microbiol. 2011, 77 (14), 4886–4893.
https://doi.org/10.1128/AEM.00091-11.
(55) Wang, Y.; Chen, G.-Q. Polyhydroxyalkanoates: Sustainability, Production, and
Industrialization. In Sustainable Polymers from Biomass; Wiley-VCH Verlag GmbH & Co.
KGaA: Weinheim, Germany, 2017; pp 11–33. https://doi.org/10.1002/9783527340200.ch2.
(56) Verhoogt, H.; Ramsay, B. A.; Favis, B. D. Polymer Blends Containing Poly(3-
Hydroxyalkanoate)S. Polymer (Guildf). 1994, 35 (24), 5155–5169.
https://doi.org/10.1016/0032-3861(94)90465-0.
(57) Sheu, D. S.; Chen, W. M.; Yang, J. Y.; Chang, R. C. Thermophilic Bacterium Caldimonas
Taiwanensis Produces Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) from Starch and
Valerate as Carbon Sources. Enzyme Microb. Technol. 2009, 44 (5), 289–294.
https://doi.org/10.1016/j.enzmictec.2009.01.004.
(58) Strong, P.; Laycock, B.; Mahamud, S.; Jensen, P.; Lant, P.; Tyson, G.; Pratt, S. The
Opportunity for High-Performance Biomaterials from Methane. Microorganisms 2016, 4
(1), 11. https://doi.org/10.3390/microorganisms4010011.
(59) Pérez-Arauz, A. O.; Aguilar-Rabiela, A. E.; Vargas-Torres, A.; Rodríguez-Hernández, A.
I.; Chavarría-Hernández, N.; Vergara-Porras, B.; López-Cuellar, M. R. Production and
Characterization of Biodegradable Films of a Novel Polyhydroxyalkanoate (PHA)
Synthesized from Peanut Oil. Food Packag. Shelf Life 2019, 20 (December 2018), 100297.
https://doi.org/10.1016/j.fpsl.2019.01.001.
(60) Kahar, P.; Tsuge, T.; Taguchi, K.; Doi, Y. High Yield Production of Polyhydroxyalkanoates
89
from Soybean Oil by Ralstonia Eutropha and Its Recombinant Strain. Polym. Degrad. Stab.
2004, 83 (1), 79–86. https://doi.org/10.1016/S0141-3910(03)00227-1.
(61) Tsuge, T.; Yamamoto, T.; Yano, K.; Abe, H.; Doi, Y.; Taguchi, S. Evaluating the Ability
of Polyhydroxyalkanoate Synthase Mutants to Produce P(3HB-Co-3HA) from Soybean Oil.
Macromol. Biosci. 2009, 9 (1), 71–78. https://doi.org/10.1002/mabi.200800118.
(62) Dennis, D.; McCoy, M.; Stangl, A.; Valentin, H. E.; Wu, Z. Formation of Poly(3-
Hydroxybutyrate-Co-3-Hydroxyhexanoate) by PHA Synthase from Ralstonia Eutropha. J.
Biotechnol. 1998, 64 (2–3), 177–186. https://doi.org/10.1016/S0168-1656(98)00110-2.
(63) Verlinden, R. A. J.; Hill, D. J.; Kenward, M. A.; Williams, C. D.; Piotrowska-Seget, Z.;
Radecka, I. K. Production of Polyhydroxyalkanoates from Waste Frying Oil by Cupriavidus
Necator. AMB Express 2011, 1 (1), 1–8. https://doi.org/10.1186/2191-0855-1-11.
(64) Morais, C.; Freitas, F.; Cruz, M. V.; Paiva, A.; Dionísio, M.; Reis, M. A. M. Conversion of
Fat-Containing Waste from the Margarine Manufacturing Process into Bacterial
Polyhydroxyalkanoates. Int. J. Biol. Macromol. 2014, 71, 68–73.
https://doi.org/10.1016/j.ijbiomac.2014.04.044.
(65) Andreeßen, B.; Steinbüchel, A. Biosynthesis and Biodegradation of 3-Hydroxypropionate-
Containing Polyesters. Appl. Environ. Microbiol. 2010, 76 (15), 4919–4925.
https://doi.org/10.1128/AEM.01015-10.
(66) Vikhoreva, G. A.; Kil’deeva, N. R.; Bonartseva, G. A.; Fedorov, M. B.; Mokhova, O. N.;
Gal’braikh, L. S. Antimicrobial Activity of Core-Sheath Surgical Sutures Modified with
Poly-3-Hydroxybutyrate. Appl. Biochem. Microbiol. 2007, 43 (6), 611–615.
https://doi.org/10.1134/s0003683807060075.
(67) Garrido, L.; Jiménez, I.; Ellis, G.; Cano, P.; García-Martínez, J. M.; López, L.; De La Peña,
E. Characterization of Surface-Modified Polyalkanoate Films for Biomedical Applications.
J. Appl. Polym. Sci. 2011, 119 (6), 3286–3296. https://doi.org/10.1002/app.32920.
90
(68) Zhao, N.; Wei, K.; Chen, J.; Zhang, S.; Wang, Y.; Wang, X. Fabrication, Characterizat ion
and Long-Term in Vitro Release of Hydrophilic Drug Using PHBV/HA Composite
Microspheres. Mater. Lett. 2006, 61 (4–5), 1071–1076.
https://doi.org/10.1016/j.matlet.2006.06.062.
(69) Paşcu, E. I.; Stokes, J.; McGuinness, G. B. Electrospun Composites of PHBV, Silk Fibroin
and Nano-Hydroxyapatite for Bone Tissue Engineering. Mater. Sci. Eng. C 2013, 33 (8),
4905–4916. https://doi.org/10.1016/j.msec.2013.08.012.
(70) Ghebi, A.; Khoshnevisan, K.; Ketabchi, N.; Derakhshan, M. A.; Babadi, A. A. Applicat ion
of Electrospun Nanofibrous PHBV Scaffold in Neural Graft and Regeneration : A Mini-
Review. Nanomed Res J 2016, 1 (2), 107–111. https://doi.org/10.7508/nmrj.2016.02.007.
(71) Plastics Europe. Biodegradable Plastics https://www.plasticseurope.org/en/about-
plastics/what-are-plastics/large-family/biodegradable-plastics (accessed Jan 13, 2020).
(72) Corbin, A.; Cowan, J.; Miles, C. A.; Hayes, D.; John, D.; Inglis, D. Using Biodegradable
Plastics as Agricultural Mulches; Washington, 2013.
(73) Dharmalingam, S. Biodegradation and Photodegradation of Polylactic Acid and Polylact ic
Acid/ Polyhydroxyalkanoate Blends Nonwoven Agricultural Mulches in Ambient Soil
Conditions, University of Tennessee, 2014.
(74) Poltronieri, P.; Kumar, P. Polyhydroxyalkanoates (PHAs) in Industrial Applications. In
Handbook of Ecomaterials; Springer International Publishing: Cham, 2018; pp 1–30.
https://doi.org/10.1007/978-3-319-48281-1_70-2.
(75) Amaro, L. P.; Abdelwahab, M. A.; Morelli, A.; Chiellini, E.; Ed, M. K. Bacterial Polyesters :
The Issue of Their Market Acceptance and Potential Solutions. In Recent Advances in
Biotechnology; Pérez Amaro, L., A. Abdelwahab, M., Morelli, A., Chiellini, F., Chiellini,
E., Eds.; BENTHAM SCIENCE PUBLISHERS, 2016; Vol. 2, pp 3–74.
https://doi.org/10.2174/9781681083735116020004.
91
(76) Raza, Z. A.; Riaz, S.; Banat, I. M. Polyhydroxyalkanoates: Properties and Chemica l
Modification Approaches for Their Functionalization. Biotechnol. Prog. 2018, 34 (1), 29–
41. https://doi.org/10.1002/btpr.2565.
(77) Chang, H. M.; Wang, Z. H.; Luo, H. N.; Xu, M.; Ren, X. Y.; Zheng, G. X.; Wu, B. J.;
Zhang, X. H.; Lu, X. Y.; Chen, F.; et al. Poly(3-Hydroxybutyrate-Co-3-
Hydroxyhexanoate)- Based Scaffolds for Tissue Engineering. Brazilian J. Med. Biol. Res.
2014, 47 (7), 533–539. https://doi.org/10.1590/1414-431X20143930.
(78) Noda, I.; Green, P. R.; Satkowski, M. M.; Schechtman, L. A. Preparation and Properties of
a Novel Class of Polyhydroxyalkanoate Copolymers. Biomacromolecules 2005, 6 (2), 580–
586. https://doi.org/10.1021/bm049472m.
(79) Changwichan, K.; Silalertruksa, T.; Gheewala, S. H. Eco-Efficiency Assessment of
Bioplastics Production Systems and End-of-Life Options. Sustain. 2018, 10 (4), 1–15.
https://doi.org/10.3390/su10040952.
(80) Gerard, T.; Budtova, T. Morphology and Molten-State Rheology of Polylactide and
Polyhydroxyalkanoate Blends. Eur. Polym. J. 2012, 48 (6), 1110–1117.
https://doi.org/10.1016/j.eurpolymj.2012.03.015.
(81) Chiono, V.; Ciardelli, G.; Vozzi, G.; Sotgiu, M. G.; Vinci, B.; Domenici, C.; Giusti, P.
Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate)/Poly(ε-Caprolactone) Blends for Tissue
Engineering Applications in the Form of Hollow Fibers. J. Biomed. Mater. Res. - Part A
2008, 85 (4), 938–953. https://doi.org/10.1002/jbm.a.31513.
(82) Ahankari, S. S.; Mohanty, A. K.; Misra, M. Mechanical Behaviour of Agro-Residue
Reinforced Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate), (PHBV) Green Composites:
A Comparison with Traditional Polypropylene Composites. Compos. Sci. Technol. 2011,
71 (5), 653–657. https://doi.org/10.1016/j.compscitech.2011.01.007.
(83) Russo, P.; Carfagna, C.; Cimino, F.; Acierno, D.; Persico, P. Biodegradable Composites
Reinforced with Kenaf Fibers: Thermal, Mechanical, and Morphological Issues. Adv.
92
Polym. Technol. 2013, 32 (SUPPL.1). https://doi.org/10.1002/adv.21282.
(84) Rudnik, E. Properties and Applications. In Compostable Polymer Materials; Elsevier, 2019;
Vol. 21, pp 49–98. https://doi.org/10.1016/B978-0-08-099438-3.00003-3.
(85) Chen, G. Q. A Microbial Polyhydroxyalkanoates (PHA) Based Bio- and Materials Industry.
Chem. Soc. Rev. 2009, 38 (8), 2434–2446. https://doi.org/10.1039/b812677c.
(86) Rastogi, V. K.; Samyn, P. Bio-Based Coatings for Paper Applications. Coatings 2015, 5
(4), 887–930. https://doi.org/10.3390/coatings5040887.
(87) Lucas, N.; Bienaime, C.; Belloy, C.; Queneudec, M.; Silvestre, F.; Nava-Saucedo, J. E.
Polymer Biodegradation: Mechanisms and Estimation Techniques - A Review.
Chemosphere 2008, 73 (4), 429–442. https://doi.org/10.1016/j.chemosphere.2008.06.064.
(88) Kijchavengkul, T.; Auras, R.; Rubino, M.; Selke, S.; Ngouajio, M.; Fernandez, R. T.
Biodegradation and Hydrolysis Rate of Aliphatic Aromatic Polyester. Polym. Degrad. Stab.
2010, 95 (12), 2641–2647. https://doi.org/10.1016/j.polymdegradstab.2010.07.018.
(89) Bugnicourt, E.; Cinelli, P.; Lazzeri, A.; Alvarez, V. The Main Characteristics, Properties,
Improvements, and Market Data of Polyhydroxyalkanoates. Handb. Sustain. Polym. 2015,
No. October, 899–928. https://doi.org/10.1201/b19600-25.
(90) Michalak, M.; Kwiecień, M.; Kawalec, M.; Kurcok, P. Oxidative Degradation of Poly(3-
Hydroxybutyrate). A New Method of Synthesis for the Malic Acid Copolymers. RSC Adv.
2016, 6 (16), 12809–12818. https://doi.org/10.1039/c5ra27041c.
(91) Aoyagi, Y.; Yamashita, K.; Doi, Y. Thermal Degradation of Poly[(R)-3-Hydroxybutyrate ],
Poly[ε-Caprolactone], and Poly[(S)-Lactide]. Polym. Degrad. Stab. 2002, 76 (1), 53–59.
https://doi.org/10.1016/S0141-3910(01)00265-8.
(92) Liu, M.; Zhuo, J. K.; Xiong, S. J.; Yao, Q. Catalytic Degradation of High-Dens ity
Polyethylene over a Clay Catalyst Compared with Other Catalysts. Energy and Fuels 2014,
28 (9), 6038–6045. https://doi.org/10.1021/ef501326k.
93
(93) Biodegradable Polymers for Industrial Applications; Smith, R., Ed.; 2005.
(94) Boopathy, R. Factors Limiting Bioremediation Technologies. Bioresour. Technol. 2000, 74
(1), 63–67. https://doi.org/10.1016/S0960-8524(99)00144-3.
(95) Jayasekara, R.; Harding, I.; Bowater, I.; Lonergan, G. Biodegradability of a Selected Range
of Polymers and Polymer Blends and Standard Methods for Assessment of Biodegradation.
J. Polym. Environ. 2005, 13 (3), 231–251. https://doi.org/10.1007/s10924-005-4758-2.
(96) Burkersroda, F. von; Schedl, L.; Göpferich, A. Why Degradable Polymers Undergo Surface
Erosion or Bulk Erosion. Biomaterials 2002, 23 (21), 4221–4231.
https://doi.org/10.1016/S0142-9612(02)00170-9.
(97) van Dijkhuizen-Radersma, R.; Moroni, L.; Apeldoorn, A. van; Zhang, Z.; Grijpma, D.
Degradable Polymers for Tissue Engineering. In Tissue Engineering; Elsevier, 2008; pp
193–221. https://doi.org/10.1016/B978-0-12-370869-4.00007-0.
(98) Anderson, J. M.; Shive, M. S. Biodegradation and Biocompatibility of PLA and PLGA
Microspheres. Adv. Drug Deliv. Rev. 2012, 64 (SUPPL.), 72–82.
https://doi.org/10.1016/j.addr.2012.09.004.
(99) Volova, T. G.; Gladyshev, M. I.; Trusova, M. Y.; Zhila, N. O. Degradation of
Polyhydroxyalkanoates in Eutrophic Reservoir. Polym. Degrad. Stab. 2007, 92 (4), 580–
586. https://doi.org/10.1016/j.polymdegradstab.2007.01.011.
(100) Muhamad, I. I.; Joon, L. K.; Noor, M. A. M. Comparing the Degradation of Poly-β-
(Hydroxybutyrate), Poly-β –(Hydroxybutyrate-Co-Valerate)(PHBV) and PHBV / Cellulo se
Triacetate Blend. Malaysian Polym. J. 2006, 1 (1), 39–46.
(101) Xu, L.; Crawford, K.; Gorman, C. B. Effects of Temperature and PH on the Degradation of
Poly(Lactic Acid) Brushes. Macromolecules 2011, 44 (12), 4777–4782.
https://doi.org/10.1021/ma2000948.
(102) Ghosh, S. K.; Pal, S.; Ray, S. Study of Microbes Having Potentiality for Biodegradation of
94
Plastics. Environ. Sci. Pollut. Res. Int. 2013, 20 (7), 4339–4355.
https://doi.org/10.1007/s11356-013-1706-x.
(103) Arutchelvi, J.; Sudhakar, M.; Arkatkar, A.; Doble, M.; Bhaduri, S.; Uppara, P. V.
Biodegradation of Polyethylene and Polypropylene. Indian J. Biotechnol. 2008, 7 (1), 9–
22.
(104) Jacquin, J.; Cheng, J.; Odobel, C.; Pandin, C.; Conan, P.; Pujo-Pay, M.; Barbe, V.;
Meistertzheim, A. L.; Ghiglione, J. F. Microbial Ecotoxicology of Marine Plastic Debris: A
Review on Colonization and Biodegradation by the “Plastisphere.” Front. Microbiol. 2019,
10 (APR), 1–16. https://doi.org/10.3389/fmicb.2019.00865.
(105) Wang, S.; Lydon, K. A.; White, E. M.; Grubbs, J. B.; Lipp, E. K.; Locklin, J.; Jambeck, J.
R. Biodegradation of Poly(3-Hydroxybutyrate- Co-3-Hydroxyhexanoate) Plastic under
Anaerobic Sludge and Aerobic Seawater Conditions: Gas Evolution and Microbia l
Diversity. Environ. Sci. Technol. 2018, 52 (10), 5700–5709.
https://doi.org/10.1021/acs.est.7b06688.
(106) Mergaert, J.; Webb, A.; Anderson, C.; Wouters, A.; Swings, J. Microbial Degradation of
Poly(3-Hydroxybutyrate) and Poly(3- Hydroxybutyrate-Co-3-Hydroxyvalerate) in Soils.
Appl. Environ. Microbiol. 1993, 59 (10), 3233–3238.
(107) Handrick, R.; Reinhardt, S.; Schultheiss, D.; Reichart, T.; Schüler, D.; Jendrossek, V.;
Jendrossek, D. Unraveling the Function of the Rhodospirillum Rubrum Activator of
Polyhydroxybutyrate (PHB) Degradation: The Activator Is a PHB-Granule-Bound Protein
(Phasin). J. Bacteriol. 2004, 186 (8), 2466–2475. https://doi.org/10.1128/JB.186.8.2466-
2475.2004.
(108) Pedersen, M. B.; Gaudu, P.; Lechardeur, D.; Petit, M.-A.; Gruss, A. Aerobic Respiration
Metabolism in Lactic Acid Bacteria and Uses in Biotechnology. Annu. Rev. Food Sci.
Technol. 2012, 3 (1), 37–58. https://doi.org/10.1146/annurev-food-022811-101255.
(109) Kleerebezem, R.; van Loosdrecht, M. C. Mixed Culture Biotechnology for Bioenergy
95
Production. Curr. Opin. Biotechnol. 2007, 18 (3), 207–212.
https://doi.org/10.1016/j.copbio.2007.05.001.
(110) Budwill, K. Anaerobic Microbial Degradation of Poly(3-Hydroxyalkanoates) with Various
Terminal Electron Acceptors. J. Environ. Polym. Degrad. 1996, 4 (2), 91–102.
https://doi.org/10.1007/BF02074870.
(111) Ishigaki, T.; Sugano, W.; Nakanishi, A.; Tateda, M.; Ike, M.; Fujita, M. The Degradability
of Biodegradable Plastics in Aerobic and Anaerobic Waste Landfill Model Reactors.
Chemosphere 2004, 54 (3), 225–233. https://doi.org/10.1016/S0045-6535(03)00750-1.
(112) Gómez, E. F.; Michel, F. C. Biodegradability of Conventional and Bio-Based Plastics and
Natural Fiber Composites during Composting, Anaerobic Digestion and Long-Term Soil
Incubation. Polym. Degrad. Stab. 2013, 98 (12), 2583–2591.
https://doi.org/10.1016/j.polymdegradstab.2013.09.018.
(113) Kelly, D. J.; Hughes, N. J.; Poole, R. K. Microaerobic Physiology: Aerobic Respiration,
Anaerobic Respiration, and Carbon Dioxide Metabolism. In Helicobacter pylori; American
Society of Microbiology, 2001; pp 113–124. https://doi.org/10.1128/9781555818005.ch10.
(114) Tanigawa, S. Biogas: Converting Waste To Energy; 2017.
(115) Themelis, N. J.; Ulloa, P. A. Methane Generation in Landfills. Renew. Energy 2007, 32 (7),
1243–1257. https://doi.org/10.1016/j.renene.2006.04.020.
(116) Rostkowski, K. H.; Criddle, C. S.; Lepech, M. D. Cradle-to-Gate Life Cycle Assessment
for a Cradle-to-Cradle Cycle: Biogas-to-Bioplastic (and Back). Environ. Sci. Technol. 2012,
46 (18), 9822–9829. https://doi.org/10.1021/es204541w.
(117) New Plastic Economy. Oxo-Degradable Plastic Packaging Is Not a Solution To Plastic
Pollution; 2017.
(118) Jakubowicz, I.; Yarahmadi, N.; Arthurson, V. Kinetics of Abiotic and Biotic Degradability
of Low-Density Polyethylene Containing Prodegradant Additives and Its Effect on the
96
Growth of Microbial Communities. Polym. Degrad. Stab. 2011, 96 (5), 919–928.
https://doi.org/10.1016/j.polymdegradstab.2011.01.031.
(119) Chiellini, E.; Corti, A.; Swift, G. Biodegradation of Thermally-Oxidized, Fragmented Low-
Density Polyethylenes. Polym. Degrad. Stab. 2003, 81 (2), 341–351.
https://doi.org/10.1016/S0141-3910(03)00105-8.
(120) ASTM International. Standard Guide for Exposing and Testing Plastics That Degrade in
the Environment by a Combination of Oxidation and Biodegradation; 2018.
https://doi.org/10.1520/D6954-04.least.
(121) Thompson, R. C.; Moore, C. J.; Saal, F. S. V.; Swan, S. H. Plastics, the Environment and
Human Health: Current Consensus and Future Trends. Philos. Trans. R. Soc. B Biol. Sci.
2009, 364 (1526), 2153–2166. https://doi.org/10.1098/rstb.2009.0053.
(122) Vinçotte. OK biodegradable WATER : Initial acceptance tests http://www.tuv-at.be/green-
marks/doc-center/ (accessed Dec 7, 2019).
(123) ASTM International. Standard Test Method for Determining Aerobic Biodegradation of
Plastic Materials in Soil; 2018. https://doi.org/10.1520/D5988-18.2.
(124) Schaiidt, L.; Detrick, F. Cellulose Decomposition in Soil Burial Beds. I. Soil Properties in
Relation to Cellulose Degradation. Appl. Microbiol. 1956, 6, 108–114.
(125) ASTM International. Standard Test Method for Determining Aerobic Biodegradation of
Plastic Materials under Controlled Composting Conditions, Incorporating Thermophilic
Temperatures; 2015. https://doi.org/10.1520/D5338-15.2.
(126) ASTM International. Standard Specification for Labeling of Plastics Designed to Be
Aerobically Composted in Municipal or Industrial Facilities; 2019.
https://doi.org/10.1520/D6400-12.2.
(127) Tuomela, M. Degradation of Lignin and Other 14 C-Labelled Compounds in Compost and
Soil with an Emphasis on White-Rot Fungi, University of Helsinki, 2002.
97
(128) ASTM International. Standard Test Method for Determining Aerobic Biodegradation of
Plastics Buried in Sandy Marine Sediment under Controlled Laboratory Conditions; 2015.
https://doi.org/10.1520/D7991-15.
(129) Leathers, T. D.; Govind, N. S.; Greene, R. V. Biodegradation of Poly ( 3-Hydroxybutyrate-
Co-3- Hydroxyvalerate ) by a Tropical Marine Bacterium, Pseudoalteromonas Sp. NRRL
B-30083. J. Polym. Environ. 2002, 8 (3), 119–124.
(130) Gonda, K. E.; Jendrossek, D.; Molitoris, H. P. Fungal Degradation of the Thermoplast ic
Polymer Poly-β-Hydroxybutyric Acid (PHB) under Simulated Deep Sea Pressure.
Hydrobiologia 2000, 426 (1–3), 173–183. https://doi.org/10.1023/A:1003971925285.
(131) Brandl, H.; Püchner, P. Biodegradation of Plastic Bottles Made from “Biopol” in an Aquatic
Ecosystem under in Situ Conditions. Biodegradation 1991, 2 (4), 237–243.
https://doi.org/10.1007/BF00114555.
(132) Sridewi, N.; Bhubalan, K.; Sudesh, K. Degradation of Commercially Important
Polyhydroxyalkanoates in Tropical Mangrove Ecosystem. Polym. Degrad. Stab. 2006, 91
(12), 2931–2940. https://doi.org/10.1016/j.polymdegradstab.2006.08.027.
(133) Badia, J. D.; Kittikorn, T.; Strömberg, E.; Santonja-Blasco, L.; Martínez-Felipe, A.; Ribes-
Greus, A.; Ek, M.; Karlsson, S. Water Absorption and Hydrothermal Performance of
PHBV/Sisal Biocomposites. Polym. Degrad. Stab. 2014, 108, 166–174.
https://doi.org/10.1016/j.polymdegradstab.2014.04.012.
(134) Harrison, J. P.; Boardman, C.; O’Callaghan, K.; Delort, A. M.; Song, J. Biodegradabil ity
Standards for Carrier Bags and Plastic Films in Aquatic Environments: A Critical Review.
R. Soc. Open Sci. 2018, 5 (5). https://doi.org/10.1098/rsos.171792.
(135) ASTM International. Standard Specification for Non-Floating Biodegradable Plastics in
the Marine; 2005. https://doi.org/10.1520/D7081-05.
(136) Vinçotte. OK biodegradable MARINE : Initial acceptance tests http://www.tuv-at.be/green-
98
marks/doc-center/ (accessed Dec 7, 2019).
(137) Puechner, P.; Mueller, W. R.; Bardtke, D. Assessing the Biodegradation Potential of
Polymers in Screening- and Long-Term Test Systems. J. Environ. Polym. Degrad. 1995, 3
(3), 133–143. https://doi.org/10.1007/BF02068464.
(138) Eriksson, O.; Finnveden, G. Energy Recovery from Waste Incineration - The Importance of
Technology Data and System Boundaries on CO2 Emissions. Energies 2017, 10 (4).
https://doi.org/10.3390/en10040539.
(139) Heitmann, A. P.; Coura Rocha, I.; de Souza, P. P.; Oliveira, L. C. A.; Patrícia, P. S.
Photoactivation of a Biodegradable Polymer (PHB): Generation of Radicals for Pollutants
Oxidation. Catal. Today 2018, No. March, 0–1.
https://doi.org/10.1016/j.cattod.2018.12.024.
(140) Imam, S. H.; Gordon, S. H.; Shogren, R. L.; Tosteson, T. R.; Govind, N. S.; Greene, R. V.
Degradation of Starch–Poly(b-Hydroxybutyrate-Co-b-Hydroxyvalerate) Bioplastics in
Tropical Coastal Waters. Appl. Environ. Microbiol. 1999, 65 (2), 431–437.
(141) Tansengco, M.; Dogma, I. Microbial Degradation of Poly-β-Hydroxybutyrate Using
Landfill Soils. Acta Biotechnol. 1999, 19 (3), 191–203.
https://doi.org/10.1002/abio.370190302.
(142) Mukai, K.; Yamada, K.; Doi, Y. Enzymatic Degradation of Poly(Hydroxyalkanoates) by a
Marine Bacterium. Polym. Degrad. Stab. 1993, 41 (1), 85–91. https://doi.org/10.1016/0141-
3910(93)90066-R.
(143) Boyandin, A. N.; Prudnikova, S. V.; Filipenko, M. L.; Khrapov, E. A.; Vasil’ev, A. D.;
Volova, T. G. Biodegradation of Polyhydroxyalkanoates by Soil Microbial Communities of
Different Structures and Detection of PHA Degrading Microorganisms. Appl. Biochem.
Microbiol. 2012, 48 (1), 28–36. https://doi.org/10.1134/S0003683812010024.
(144) Gilmore, D. F.; Antoun, S.; Lenz, R. W.; Goodwin, S.; Austin, R.; Fuller, R. C. The Fate of
99
“biodegradable” Plastics in Municipal Leaf Compost. J. Ind. Microbiol. 1992, 10 (3–4),
199–206. https://doi.org/10.1007/BF01569767.
(145) Volova, T. G.; Boyandin, A. N.; Vasil’ev, A. D.; Karpov, V. A.; Kozhevnikov, I. V.;
Prudnikova, S. V.; Rudnev, V. P.; Xuån, B. B.; Dũng, V. V.; Gitel’zon, I. I. Biodegradation
of Polyhydroxyalkanoates (PHAs) in the South China Sea and Identification of PHA-
Degrading Bacteria. Microbiology 2011, 80 (2), 252–260.
https://doi.org/10.1134/S0026261711020184.
(146) Doi, Y.; Kanesawa, Y.; Tanahashi, N.; Kumagai, Y. Biodegradation of Microbial Polyesters
in the Marine Environment. Polym. Degrad. Stab. 1992, 36 (2), 173–177.
https://doi.org/10.1016/0141-3910(92)90154-W.
(147) Dunja-Manal, A.-Z.; Rolf-Joachim, M.; Wolf-Dieter, D. Degradation of Natural and
Synthetic Polyesters under Anaerobic Conditions. J. Biotechnol. 2001, 86 (2), 113–126.
(148) Yagi, H.; Ninomiya, F.; Funabashi, M.; Kunioka, M. Mesophilic Anaerobic Biodegradation
Test and Analysis of Eubacteria and Archaea Involved in Anaerobic Biodegradation of Four
Specified Biodegradable Polyesters. Polym. Degrad. Stab. 2014, 110, 278–283.
https://doi.org/10.1016/j.polymdegradstab.2014.08.031.
(149) Chen, H. Assessment of Biodegradation in Different Environmental Compartments of
Blends and Composites Based on Microbial Poly ( Hydroxyalkanoate )S, University of Pisa,
2012.
(150) Boyandin, A. N.; Prudnikova, S. V.; Karpov, V. A.; Ivonin, V. N.; Dỗ, N. L.; Nguyễn, T.
H.; Lê, T. M. H.; Filichev, N. L.; Levin, A. L.; Filipenko, M. L.; et al. Microbial Degradation
of Polyhydroxyalkanoates in Tropical Soils. Int. Biodeterior. Biodegrad. 2013, 83, 77–84.
https://doi.org/10.1016/j.ibiod.2013.04.014.
(151) Sang, B. I.; Hori, K.; Tanji, Y.; Unno, H. Fungal Contribution to in Situ Biodegradation of
Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) Film in Soil. Appl. Microbiol. Biotechnol.
2002, 58 (2), 241–247. https://doi.org/10.1007/s00253-001-0884-5.
100
(152) Kim, D. Y.; Rhee, Y. H. Biodegradation of Microbial and Synthetic Polyesters by Fungi.
Appl. Microbiol. Biotechnol. 2003, 61 (4), 300–308. https://doi.org/10.1007/s00253-002-
1205-3.
(153) Volova, T. G.; Boyandin, A. N.; Vasiliev, A. D.; Karpov, V. A.; Prudnikova, S. V.;
Mishukova, O. V.; Boyarskikh, U. A.; Filipenko, M. L.; Rudnev, V. P.; Bá Xuân, B.; et al.
Biodegradation of Polyhydroxyalkanoates (PHAs) in Tropical Coastal Waters and
Identification of PHA-Degrading Bacteria. Polym. Degrad. Stab. 2010, 95 (12), 2350–2359.
https://doi.org/10.1016/j.polymdegradstab.2010.08.023.
(154) Zwietering, M. H.; Jongenburger, I.; Rombouts, F. M.; Van’t Riet, K. Modeling of the
Bacterial Growth Curve. Appl. Environ. Microbiol. 1990, 56 (6), 1875–1881.
(155) Kobayashi, T.; Uchino, K.; Abe, T.; Yamazaki, Y.; Saito, T. Novel Intracellular 3-
Hydroxybutyrate-Oligomer Hydrolase in Wautersia Eutropha H16. J. Bacteriol. 2005, 187
(15), 5129–5135. https://doi.org/10.1128/JB.187.15.5129-5135.2005.
(156) Numata, K.; Abe, H.; Doi, Y. Enzymatic Processes for Biodegradation of
Poly(Hydroxyalkanoate)s Crystals. Can. J. Chem. 2008, 86 (6), 471–483.
https://doi.org/10.1139/v08-004.
(157) Scherer, T. M.; Fuller, R. C.; Lenz, R. W.; Goodwin, S. Hydrolase Activity of an
Extracellular Depolymerase from Aspergillus Fumigatus with Bacterial and Synthet ic
Polyesters. Polym. Degrad. Stab. 1999, 64 (2), 267–275. https://doi.org/10.1016/S0141-
3910(98)00201-8.
(158) Slepecky, R. A.; Law, J. H. Synthesis and Degradation of Poly-Beta-Hydroxybutyric Acid
in Connection with Sporulation of Bacillus Megaterium. J. Bacteriol. 1961, 82 (1), 37–42.
(159) Shang, L.; Fei, Q.; Zhang, Y. H.; Wang, X. Z.; Fan, D. Di; Chang, H. N. Thermal Properties
and Biodegradability Studies of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate). J.
Polym. Environ. 2012, 20 (1), 23–28. https://doi.org/10.1007/s10924-011-0362-9.
101
(160) Eldsäter, C.; Albertsson, A. C.; Karlsson, S. Impact of Degradation Mechanisms on Poly(3-
Hydroxybutyrate-Co-3-Hydroxyvalerate) during Composting. Acta Polym. 1997, 48 (11),
478–483. https://doi.org/10.1002/actp.1997.010481103.
(161) Hakkarainen, M. Aliphatic Polyesters: Abiotic and Biotic Degradation and Degradation
Products. Adv. Polym. Sci. 2002, 157, 113–138. https://doi.org/10.1007/3-540-45734-8_4.
(162) Iwata, T.; Doi, Y. Crystal Structure and Biodegradation of Aliphatic Polyester Crystals.
Macromol. Chem. Phys. 1999, 200 (11), 2429–2442. https://doi.org/10.1002/(SICI)1521-
3935(19991101)200:11<2429::AID-MACP2429>3.0.CO;2-#.
(163) Thellen, C.; Coyne, M.; Froio, D.; Auerbach, M.; Wirsen, C.; Ratto, J. A. A Processing,
Characterization and Marine Biodegradation Study of Melt-Extruded
Polyhydroxyalkanoate (PHA) Films. J. Polym. Environ. 2008, 16 (1), 1–11.
https://doi.org/10.1007/s10924-008-0079-6.
(164) Wu, C. S. Assessing Biodegradability and Mechanical, Thermal, and Morphologica l
Properties of an Acrylic Acid-Modified Poly(3-Hydroxybutyric Acid)/Wood Flours
Biocomposite. J. Appl. Polym. Sci. 2006, 102 (4), 3565–3574.
https://doi.org/10.1002/app.24817.
(165) Numata, K.; Kikkawa, Y.; Tsuge, T.; Iwata, T.; Doi, Y.; Abe, 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.
Biomacromolecules 2005, 6 (4), 2008–2016. https://doi.org/10.1021/bm0501151.
(166) Holland, S. J.; Jolly, A. M.; Yasin, M.; Tighe, B. J. Polymers for Biodegradable Medical
Devices: II. Hydroxybuyrate-Hydroxyvalerate Copolymers: Hydrolytic Degradation
Studies. Biomaterials 1987, 8, 289–295.
(167) Feng, L.; Wang, Y.; Inagawa, Y.; Kasuya, K.; Saito, T.; Doi, Y.; Inoue, Y. Enzymatic
Degradation Behavior of Comonomer Compositionally Fractionated Bacterial Poly(3-
102
Hydroxybutyrate-Co-3-Hydroxyvalerate)s by Poly(3-Hydroxyalkanoate) Depolymerases
Isolated from Ralstonia Pickettii T1 and Acidovorax Sp. TP4. Polym. Degrad. Stab. 2004,
84 (1), 95–104. https://doi.org/10.1016/j.polymdegradstab.2003.09.016.
(168) Huang, Y.; Zhang, C.; Pan, Y.; Zhou, Y.; Jiang, L.; Dan, Y. Effect of NR on the Hydrolyt ic
Degradation of PLA. Polym. Degrad. Stab. 2013, 98 (5), 943–950.
https://doi.org/10.1016/j.polymdegradstab.2013.02.018.
(169) Deroiné, M.; Le Duigou, A.; Corre, Y. M.; Le Gac, P. Y.; Davies, P.; César, G.; Bruzaud,
S. Seawater Accelerated Ageing of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate ).
Polym. Degrad. Stab. 2014, 105 (1), 237–247.
https://doi.org/10.1016/j.polymdegradstab.2014.04.026.
(170) Arcos-Hernandez, M. V.; Laycock, B.; Pratt, S.; Donose, B. C.; Nikolič, M. A. L.;
Luckman, P.; Werker, A.; Lant, P. A. Biodegradation in a Soil Environment of Activated
Sludge Derived Polyhydroxyalkanoate (PHBV). Polym. Degrad. Stab. 2012, 97 (11), 2301–
2312. https://doi.org/10.1016/j.polymdegradstab.2012.07.035.
(171) Keen, I.; Raggatt, L. J.; Cool, S. M.; Nurcombe, V.; Fredericks, P.; Trau, M.; Grøndahl, L.
Investigations into Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) Surface Properties
Causing Delayed Osteoblast Growth. J. Biomater. Sci. Polym. Ed. 2007, 18 (9), 1101–1123.
https://doi.org/10.1163/156856207781554046.
(172) Morse, M. C.; Liao, Q.; Criddle, C. S.; Frank, C. W. Anaerobic Biodegradation of the
Microbial Copolymer Poly(3-Hydroxybutyrate- Co-3-Hydroxyhexanoate): Effects of
Comonomer Content, Processing History, and Semi-Crystalline Morphology. Polymer
(Guildf). 2011, 52 (2), 547–556. https://doi.org/10.1016/j.polymer.2010.11.024.
(173) Avella, M.; La Rota, G.; Martuscelli, E.; Raimo, M.; Sadocco, P.; Elegir, G.; Riva, R.
Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) and Wheat Straw Fibre Composites:
Thermal, Mechanical Properties and Biodegradation Behaviour. J. Mater. Sci. 2000, 35 (4),
829–836. https://doi.org/10.1023/A:1004773603516.
103
(174) Muniyasamy, S.; Ofosu, O.; John, M. J.; Anandjiwala, R. D. Mineralization of Poly(Lactic
Acid) (PLA), Poly(3-Hydroxybutyrate-Co-Valerate) (PHBV) and PLA/PHBV Blend in
Compost and Soil Environments. J. Renew. Mater. 2016, 4 (2), 133–145.
https://doi.org/10.7569/JRM.2016.634104.
(175) Deroiné, M.; César, G.; Le Duigou, A.; Davies, P.; Bruzaud, S. Natural Degradation and
Biodegradation of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) in Liquid and Solid
Marine Environments. J. Polym. Environ. 2015, 23 (4), 493–505.
https://doi.org/10.1007/s10924-015-0736-5.
(176) Weng, Y. X.; Wang, Y.; Wang, X. L.; Wang, Y. Z. Biodegradation Behavior of PHBV
Films in a Pilot-Scale Composting Condition. Polym. Test. 2010, 29 (5), 579–587.
https://doi.org/10.1016/j.polymertesting.2010.04.002.
(177) Weng, Y. X.; Wang, X. L.; Wang, Y. Z. Biodegradation Behavior of PHAs with Different
Chemical Structures under Controlled Composting Conditions. Polym. Test. 2011, 30 (4),
372–380. https://doi.org/10.1016/j.polymertesting.2011.02.001.
(178) Mergaert, J.; Anderson, C.; Wouters, A.; Swings, J.; Kersters, K. Biodegradation of
Polyhydroxyalkanoates. FEMS Microbiol. Lett. 1992, 103 (2–4), 317–321.
https://doi.org/10.1016/0378-1097(92)90325-I.
(179) Volova, T. G.; Zhila, N. O.; Shishatskaya, E. I.; Mironov, P. V.; Vasil’Ev, A. D.; Sukovatyi,
A. G.; Sinskey, A. J. The Physicochemical Properties of Polyhydroxyalkanoates with
Different Chemical Structures. Polym. Sci. - Ser. A 2013, 55 (7), 427–437.
https://doi.org/10.1134/S0965545X13070080.
(180) Volova, T. G.; Prudnikova, S. V.; Vinogradova, O. N.; Syrvacheva, D. A.; Shishatskaya, E.
I. Microbial Degradation of Polyhydroxyalkanoates with Different Chemical Compositions
and Their Biodegradability. Microb. Ecol. 2017, 73 (2), 353–367.
https://doi.org/10.1007/s00248-016-0852-3.
(181) Federle, T. W.; Barlaz, M. A.; Pettigrew, C. A.; Kerr, K. M.; Kemper, J. J.; Nuck, B. A.;
104
Schechtman, L. A. Anaerobic Biodegradation of Aliphatic Polyesters: Poly(3-
Hydroxybutyrate-Co-3-Hydroxyoctanoate) and Poly(ε-Caprolactone). Biomacromolecules
2002, 3 (4), 813–822. https://doi.org/10.1021/bm025520w.
(182) Jendrossek, D. Peculiarities of PHA Granules Preparation and PHA Depolymerase Activity
Determination. Appl. Microbiol. Biotechnol. 2007, 74 (6), 1186–1196.
https://doi.org/10.1007/s00253-007-0860-9.
(183) Miyazaki, S.; Takahashi, K.; Shiraki, M.; Saito, T.; Tezuka, Y.; Kasuya, K. I. Properties of
a Poly(3-Hydroxybutyrate) Depolymerase from Penicillium Funiculosum. J. Polym.
Environ. 2000, 8 (4), 175–182. https://doi.org/10.1023/A:1015245710406.
(184) Martínez, V.; de Santos, P. G.; García-Hidalgo, J.; Hormigo, D.; Prieto, M. A.; Arroyo, M.;
de la Mata, I. Novel Extracellular Medium-Chain-Length Polyhydroxyalkanoa te
Depolymerase from Streptomyces Exfoliatus K10 DSMZ 41693: A Promising Biocatalys t
for the Efficient Degradation of Natural and Functionalized Mcl-PHAs. Appl. Microbiol.
Biotechnol. 2015, 99 (22), 9605–9615. https://doi.org/10.1007/s00253-015-6780-1.
(185) Mergaert, J.; Swings, J. Biodiversity of Microorganisms That Degrade Bacterial and
Synthetic Polyesters. J. Ind. Microbiol. Biotechnol. 1996, 17 (5–6), 463–469.
https://doi.org/10.1007/bf01574777.
(186) Siemann, U. Solvent Cast Technology – a Versatile Tool for Thin Film Production. In
Scattering Methods and the Properties of Polymer Materials; Springer Berlin Heidelberg:
Berlin, Heidelberg, 2005; Vol. 130, pp 1–14. https://doi.org/10.1007/b107336.
(187) Kasuya, K.; Takagi, K.; Ishiwatari, S.; Yoshida, Y.; Doi, Y. Biodegradabilities of Various
Aliphatic Polyesters in Natural Waters. Polym. Degrad. Stab. 2002, 59 (1–3), 327–332.
https://doi.org/10.1016/s0141-3910(97)00155-9.
(188) Gutierrez-Wing, M. T.; Stevens, B. E.; Theegala, C. S.; Negulescu, I. I.; Rusch, K. A.
Anaerobic Biodegradation of Polyhydroxybutyrate in Municipal Sewage Sludge. J.
Environ. Eng. 2010, 136 (7), 709–718. https://doi.org/10.1061/(ASCE)EE.1943-
105
7870.0000208.
(189) Sashiwa, H.; Fukuda, R.; Okura, T.; Sato, S.; Nakayama, A. Microbial Degradation
Behavior in Seawater of Polyester Blends Containing Poly(3-Hydroxybutyrate-Co-3-
Hydroxyhexanoate) (PHBHHx). Mar. Drugs 2018, 16 (1), 1–11.
https://doi.org/10.3390/md16010034.
(190) Wu, C. S. Preparation, Characterization and Biodegradability of Crosslinked Tea Plant -
Fibre-Reinforced Polyhydroxyalkanoate Composites. Polym. Degrad. Stab. 2013, 98 (8),
1473–1480. https://doi.org/10.1016/j.polymdegradstab.2013.04.013.
(191) Wu, C. S.; Liao, H. T.; Cai, Y. X. Characterisation, Biodegradability and Application of
Palm Fibre-Reinforced Polyhydroxyalkanoate Composites. Polym. Degrad. Stab. 2017,
140, 55–63. https://doi.org/10.1016/j.polymdegradstab.2017.04.016.
(192) Altaee, N.; El-Hiti, G. A.; Fahdil, A.; Sudesh, K.; Yousif, E. Biodegradation of Different
Formulations of Polyhydroxybutyrate Films in Soil. Springerplus 2016, 5 (1).
https://doi.org/10.1186/s40064-016-2480-2.
(193) Seggiani, M.; Cinelli, P.; Verstichel, S.; Puccini, M.; Vitolo, S.; Anguillesi, I.; Lazzeri, A.
Development of Fibres-Reinforced Biodegradable Composites. Chem. Eng. Trans. 2015,
43, 1813–1818. https://doi.org/10.3303/CET1543303.
(194) Yoon, J. S.; Chang, M. C.; Kim, M. N.; Kang, E. J.; Kim, C.; Chin, I. J. Compatibility and
Fungal Degradation of Poly[(R)-3-Hydroxybutyrate]/Aliphatic Copolyester Blend. J.
Polym. Sci. Part B Polym. Phys. 1996, 34 (15), 2543–2551.
https://doi.org/10.1002/(SICI)1099-0488(19961115)34:15<2543::AID-POLB2>3.0.CO;2-
U.
(195) Woolnough, C. A.; Yee, L. H.; Charlton, T. S.; Foster, L. J. R. A Tuneable Switch for
Controlling Environmental Degradation of Bioplastics: Addition of Isothiazolinone to
Polyhydroxyalkanoates. PLoS One 2013, 8 (10), 1–10.
https://doi.org/10.1371/journal.pone.0075817.
106
(196) Duangphet, S.; Szegda, D.; Song, J.; Tarverdi, K. The Effect of Chain Extender on Poly(3-
Hydroxybutyrate-Co-3-Hydroxyvalerate): Thermal Degradation, Crystallization, and
Rheological Behaviours. J. Polym. Environ. 2014, 22 (1), 1–8.
https://doi.org/10.1007/s10924-012-0568-5.
(197) Freitas, A. L. P. de L.; Tonini Filho, L. R.; Calvão, P. S.; Souza, A. M. C. de. Effect of
Montmorillonite and Chain Extender on Rheological, Morphological and Biodegradation
Behavior of PLA/PBAT Blends. Polym. Test. 2017, 62, 189–195.
https://doi.org/10.1016/j.polymertesting.2017.06.030.
(198) Dong, W.; Zou, B.; Yan, Y.; Ma, P.; Chen, M. Effect of Chain-Extenders on the Properties
and Hydrolytic Degradation Behavior of the Poly(Lactide)/ Poly(Butylene Adipate-Co-
Terephthalate) Blends. Int. J. Mol. Sci. 2013, 14 (10), 20189–20203.
https://doi.org/10.3390/ijms141020189.
(199) Savenkova, L.; Gercberga, Z.; Nikolaeva, V.; Dzene, A.; Bibers, I.; Kalnin, M. Mechanica l
Properties and Biodegradation Characteristics of PHB-Based Films. Process Biochem.
2000, 35 (6), 573–579. https://doi.org/10.1016/S0032-9592(99)00107-7.
(200) Fonseca, J. D.; Latifi, A. M.; Orjuela, A.; Gil, I. D.; Rodríguez, G. Dynamic Simulation and
Optimisation of an Industrial Process for Tributyl Citrate Production; Elsevier Masson
SAS, 2016; Vol. 38. https://doi.org/10.1016/B978-0-444-63428-3.50194-6.
(201) Râpə, M.; Darie-Nitə, R. N.; Grosu, E.; Tənase, E. E.; Trifoi, A. R.; Pap, T.; Vasile, C.
Effect of Plasticizers on Melt Processability and Properties of PHB. J. Optoelectron. Adv.
Mater. 2015, 17 (11–12), 1778–1784.
(202) Höglund, A.; Hakkarainen, M.; Albertsson, A. C. Migration and Hydrolysis of Hydrophobic
Polylactide Plasticizer. Biomacromolecules 2010, 11 (1), 277–283.
https://doi.org/10.1021/bm901157h.
(203) Kranz, H.; Ubrich, N.; Maincent, P.; Bodmeier, R. Physicomechanical Properties of
Biodegradable Poly(D,L-Lactide) and Poly(D,L-Lactide-Co-Glycolide) Films in the Dry
107
and Wet States. J. Pharm. Sci. 2000, 89 (12), 1558–1566. https://doi.org/10.1002/1520-
6017(200012)89:12<1558::AID-JPS6>3.0.CO;2-8.
(204) Teramoto, N.; Urata, K.; Ozawa, K.; Shibata, M. Biodegradation of Aliphatic Polyester
Composites Reinforced by Abaca Fiber. Polym. Degrad. Stab. 2004, 86 (3), 401–409.
https://doi.org/10.1016/j.polymdegradstab.2004.04.026.
(205) Abdelwahab, M. A.; Flynn, A.; Chiou, B. Sen; Imam, S.; Orts, W.; Chiellini, E. Thermal,
Mechanical and Morphological Characterization of Plasticized PLA-PHB Blends. Polym.
Degrad. Stab. 2012, 97 (9), 1822–1828.
https://doi.org/10.1016/j.polymdegradstab.2012.05.036.
(206) Weng, Y. X.; Wang, L.; Zhang, M.; Wang, X. L.; Wang, Y. Z. Biodegradation Behavior of
P(3HB,4HB)/PLA Blends in Real Soil Environments. Polym. Test. 2013, 32 (1), 60–70.
https://doi.org/10.1016/j.polymertesting.2012.09.014.
(207) Wang, J.; Mao, Q. Methodology Based on the PVT Behavior of Polymer for Injection
Molding. Adv. Polym. Technol. 2012, 32 (2013), 474–485. https://doi.org/10.1002/adv.
(208) Redondo, D.; Peñalva, C.; Val, J.; Braca, F.; Pérez, M. Biodegradable Plastics for Improving
Soil and Fruit Quality Characteristics. Acta Hortic. 2019, 1252, 69–76.
https://doi.org/10.17660/ActaHortic.2019.1252.9.
(209) Sato, H.; Murakami, R.; Padermshoke, A.; Hirose, F.; Senda, K.; Noda, I.; Ozaki, Y.
Infrared Spectroscopy Studies of CH⋯O Hydrogen Bondings and Thermal Behavior of
Biodegradable Poly(Hydroxyalkanoate). Macromolecules 2004, 37 (19), 7203–7213.
https://doi.org/10.1021/ma049117o.
(210) Hegde, S.; Dell, E.; Lewis, C.; Trabold, T. A.; Diaz, C. A. Anaerobic Biodegradation of
Bioplastic Packaging Materials. 21st IAPRI World Conf. Packag. 2018 - Packag. Driv. a
Sustain. Futur. 2019, 730–737. https://doi.org/10.12783/iapri2018/24453.
(211) Arrieta, M. P.; López, J.; Rayón, E.; Jiménez, A. Disintegrability under Composting
108
Conditions of Plasticized PLA–PHB Blends. Polym. Degrad. Stab. 2014, 108, 307–318.
https://doi.org/10.1016/j.polymdegradstab.2014.01.034.
(212) Modelli, A.; Calcagno, B.; Scandola, M. Kinetics of Aerobic Polymer Degradation in Soil
by Means of the ASTM D 5988-96 Standard Method. J. Environ. Polym. Degrad. 1999, 7
(2), 109–116. https://doi.org/10.1023/a:1021864402395.
(213) Shibata, M.; Oyamada, S.; Kobayashi, S.; Yaginuma, D. Mechanical Properties and
Biodegradability of Green Composites Based on Biodegradable Polyesters and Lyocell
Fabric. J. Appl. Polym. Sci. 2004, 92 (6), 3857–3863. https://doi.org/10.1002/app.20405.
(214) Vinçotte. OK Biodegradable SOIL : Initial Acceptance Tests; 2012.
(215) Wu, C. S. Preparation and Characterization of Polyhydroxyalkanoate Bioplastic-Based
Green Renewable Composites from Rice Husk. J. Polym. Environ. 2014, 22 (3), 384–392.
https://doi.org/10.1007/s10924-014-0662-y.
(216) Wu, C. S. Characterization and Biodegradation Evaluation of Biocapsules Composed of
Polyester/Natural Product Composites. Polym. - Plast. Technol. Eng. 2016, 55 (4), 391–
402. https://doi.org/10.1080/03602559.2015.1098683.
(217) Luzier, W. D. Materials Derived from Biomass/Biodegradable Materials. Proc. Natl. Acad.
Sci. U. S. A. 1992, 89 (3), 839–842.
(218) Batista, K. C.; Silva, D. A. K.; Coelho, L. A. F.; Pezzin, S. H.; Pezzin, A. P. T. Soil
Biodegradation of PHBV/Peach Palm Particles Biocomposites. J. Polym. Environ. 2010, 18
(3), 346–354. https://doi.org/10.1007/s10924-010-0238-4.
(219) Madbouly, S. A.; Schrader, J. A.; Srinivasan, G.; Liu, K.; McCabe, K. G.; Grewell, D.;
Graves, W. R.; Kessler, M. R. Biodegradation Behavior of Bacterial-Based
Polyhydroxyalkanoate (PHA) and DDGS Composites. Green Chem. 2014, 16 (4), 1911–
1920. https://doi.org/10.1039/c3gc41503a.
(220) Joyyi, L.; Ahmad Thirmizir, M. Z.; Salim, M. S.; Han, L.; Murugan, P.; Kasuya, K. ichi;
109
Maurer, F. H. J.; Zainal Arifin, M. I.; Sudesh, K. Composite Properties and Biodegradation
of Biologically Recovered P(3HB-Co-3HHx) Reinforced with Short Kenaf Fibers. Polym.
Degrad. Stab. 2017, 137, 100–108.
https://doi.org/10.1016/j.polymdegradstab.2017.01.004.
(221) Chan, C. M.; Vandi, L. J.; Pratt, S.; Halley, P.; Richardson, D.; Werker, A.; Laycock, B.
Insights into the Biodegradation of PHA / Wood Composites: Micro- and Macroscopic
Changes. Sustain. Mater. Technol. 2019, 21, e00099.
https://doi.org/10.1016/j.susmat.2019.e00099.
(222) Celestina, C.; Wood, J. L.; Manson, J. B.; Wang, X.; Sale, P. W. G.; Tang, C.; Franks, A.
E. Microbial Communities in Top- and Subsoil of Repacked Soil Columns Respond
Differently to Amendments but Their Diversity Is Negatively Correlated with Plant
Productivity. Sci. Rep. 2019, 9 (1), 1–12. https://doi.org/10.1038/s41598-019-45368-9.
(223) Sayyed, R. Z.; Wani, S. J.; Alyousef, A. A.; Alqasim, A.; Syed, A.; El-Enshasy, H. A.
Purification and Kinetics of the PHB Depolymerase of Microbacterium Paraoxydans RZS6
Isolated from a Dumping Yard. PLoS One 2019, 14 (6), 1–14.
https://doi.org/10.1371/journal.pone.0212324.
(224) Manna, A.; Paul, A. K. Degradation of Microbial Polyester Poly(3-Hydroxybutyrate) in
Environmental Samples and in Culture. Biodegradation 2000, 11 (5), 323–329.
https://doi.org/10.1023/A:1011162624704.
(225) Tsuji, H.; Suzuyoshi, K. Environmental Degradation of Biodegradable Polyesters. IV. The
Effects of Pores and Surface Hydrophilicity on the Biodegradation of Poly(Εcaprolactone)
and Poly[(R)-3-Hydroxybutyrate] Films in Controlled Seawater. J. Appl. Polym. Sci. 2003,
90 (2), 587–593. https://doi.org/10.1002/app.12781.
(226) Slessarev, E. W.; Lin, Y.; Bingham, N. L.; Johnson, J. E.; Dai, Y.; Schimel, J. P.; Chadwick,
O. A. Water Balance Creates a Threshold in Soil PH at the Global Scale. Nature 2016, 540
(7634), 567–569. https://doi.org/10.1038/nature20139.
110
(227) Kim, M.; Lee, A.; Yoon, J.; Chin, I. Biodegradation of Poly(3-Hydroxybutyrate), Sky-
Green® and Mater-Bi® by Fungi Isolated from Soils. Eur. Polym. J. 2000, 36 (8), 1677–
1685. https://doi.org/10.1016/S0014-3057(99)00219-0.
(228) Rudnik, E.; Briassoulis, D. Comparative Biodegradation in Soil Behaviour of Two
Biodegradable Polymers Based on Renewable Resources. J. Polym. Environ. 2011, 19 (1),
18–39. https://doi.org/10.1007/s10924-010-0243-7.
(229) Rosa, D. S.; Filho, R. P.; Chui, Q. S. H.; Calil, M. R.; Guedes, C. G. F. The Biodegradation
of Poly- β -(Hydroxybutyrate), Poly- β -(Hydroxybutyrate-Co- β -Valerate) and Poly(-
Caprolactone) in Compost Derived from Municipal Solid Waste. Eur. Polym. J. 2003, 39
(2), 233–237. https://doi.org/10.1016/S0014-3057(02)00215-X.
(230) Gunning, M. A.; Geever, L. M.; Killion, J. A.; Lyons, J. G.; Higginbotham, C. L.
Mechanical and Biodegradation Performance of Short Natural Fibre Polyhydroxybutyra te
Composites. Polym. Test. 2013, 32 (8), 1603–1611.
https://doi.org/10.1016/j.polymertesting.2013.10.011.
(231) Rutkowska, M.; Krasowska, K.; Heimowska, A.; Adamus, G.; Sobota, M.; Musioł, M.;
Janeczek, H.; Sikorska, W.; Krzan, A.; Žagar, E.; et al. Environmental Degradation of
Blends of Atactic Poly[(R,S)-3-Hydroxybutyrate] with Natural PHBV in Baltic Sea Water
and Compost with Activated Sludge. J. Polym. Environ. 2008, 16 (3), 183–191.
https://doi.org/10.1007/s10924-008-0100-0.
(232) Luo, S.; Netravali, A. N. A Study of Physical and Mechanical Properties of
Poly(Hydroxybutyrate-Co-Hydroxyvalerate) during Composting. Polym. Degrad. Stab.
2003, 80 (1), 59–66. https://doi.org/10.1016/S0141-3910(02)00383-X.
(233) Mergaert, J.; Anderson, C.; Wouters, A.; Swings, J. Microbial Degradation of Poly(3-
Hydroxybutyrate) and Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) in Compost. J.
Environ. Polym. Degrad. 1994, 2 (3), 177–183. https://doi.org/10.1007/BF02067443.
(234) Greene, J. PLA and PHA Biodegradation in the Marine Environment; Chico, 2012.
111
(235) Ho, Y. H.; Gan, S. N.; Tan, I. K. P. Biodegradation of a Medium-Chain-Length
Polyhydroxyalkanoate in Tropical River Water. Appl. Biochem. Biotechnol. - Part A Enzym.
Eng. Biotechnol. 2002, 102–103, 337–347. https://doi.org/10.1385/ABAB:102-103 :1-
6:337.
(236) Halliday, E.; McLellan, S. L.; Amaral-Zettler, L. A.; Sogin, M. L.; Gast, R. J. Comparison
of Bacterial Communities in Sands and Water at Beaches with Bacterial Water Quality
Violations. PLoS One 2014, 9 (3). https://doi.org/10.1371/journal.pone.0090815.
(237) Ramsay, B. A.; Langlade, V.; Carreau, P. J.; Ramsay, J. A. Biodegradability and
Mechanical Properties of Poly-(β-Hydroxybutyrate- Co-β-Hydroxyvalerate)-Starch Blends.
Appl. Environ. Microbiol. 1993, 59 (4), 1242–1246.
(238) Mergaert, J.; Wouters, A.; Anderson, C.; Swings, J. In Situ Biodegradation of Poly(3-
Hydroxybutyrate) and Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) in Natural Waters.
Can. J. Microbiol. 1995, 41 (SUPPL. 1), 154–159. https://doi.org/10.1139/m95-182.
(239) Tsuji, H.; Suzuyoshi, K. Environmental Degradation of Biodegradable Polyesters and Poly
( L-Lactide ) Films in Natural Dynamic Seawater. Polym. Degrad. Stab. 2002, 75, 357–365.
(240) Shin, P. K.; Kirn, M. H.; Kim, J. M. Biodegradability of Degradable Plastics Exposed to
Anaerobic Digested Sludge and Simulated Landfill Conditions. J. Environ. Polym. Degrad.
1997, 5 (1), 33–39. https://doi.org/10.1007/BF02763566.
(241) Yagi, H.; Ninomiya, F.; Funabashi, M.; Kunioka, M. Thermophilic Anaerobic
Biodegradation Test and Analysis of Eubacteria Involved in Anaerobic Biodegradation of
Four Specified Biodegradable Polyesters. Polym. Degrad. Stab. 2013, 98 (6), 1182–1187.
https://doi.org/10.1016/j.polymdegradstab.2013.03.010.
(242) Benn, N.; Zitomer, D. Pretreatment and Anaerobic Co-Digestion of Selected PHB and PLA
Bioplastics. Front. Environ. Sci. 2018, 5 (JAN), 1–9.
https://doi.org/10.3389/fenvs.2017.00093.
112
(243) Labatut, R. A.; Angenent, L. T.; Scott, N. R. Conventional Mesophilic vs . Thermophilic
Anaerobic Digestion : A Trade-off between Performance and Stability ? Water Res. 2014,
53, 249–258. https://doi.org/10.1016/j.watres.2014.01.035.
(244) El-mashad, H. M.; Zhang, R.; Greene, J. P. Anaerobic Biodegradability of Selected
Biodegradable Plastics and Biobased Products. J. Environ. Sci. Eng. A 2012, 1 (January
2012), 108–114.
(245) Weaver, J. E. Effect of Inoculum Source on the Rate and Extent of Anaerobic
Biodegradation, North Carolina State University, 2013.
(246) Green Biorenewable Biocomposites; Thakur, V. K., Kessler, M. R., Eds.; Apple Academic
Press, 2016. https://doi.org/10.1201/b18092.
(247) Monti, A.; Alexopoulou, E. Kenaf: A Multi-Purpose Crop for Several Industria l
Applications: New Insights from the Biokenaf Project. In Green Energy and Technology;
2013; Vol. 117. https://doi.org/10.1007/978-1-4471-5067-1.
(248) Cazaurang-Martinez, M. N.; Herrera-Franco, P. J.; Gonzalez-Chi, P. I.; Aguilar-Vega, M.
Physical and Mechanical Properties of Henequen Fibers. J. Appl. Polym. Sci. 1991, 43 (4),
749–756. https://doi.org/10.1002/app.1991.070430412.
(249) Shibata, M.; Takachiyo, K. I.; Ozawa, K.; Yosomiya, R.; Takeishi, H. Biodegradab le
Polyester Composites Reinforced with Short Abaca Fiber. J. Appl. Polym. Sci. 2002, 85 (1),
129–138. https://doi.org/10.1002/app.10665.
(250) Shanks, R. A.; Hodzic, A.; Wong, S. Thermoplastic Biopolyester Natural Fiber Composites.
J. Appl. Polym. Sci. 2004, 91 (4), 2114–2121. https://doi.org/10.1002/app.13289.
(251) Moliner, C.; Badia, J. D.; Bosio, B.; Arato, E.; Kittikorn, T.; Strömberg, E.; Teruel-Juanes,
R.; Ek, M.; Karlsson, S.; Ribes-Greus, A. Thermal and Thermo-Oxidative Stability and
Kinetics of Decomposition of PHBV/Sisal Composites. Chem. Eng. Commun. 2018, 205
(2), 226–237. https://doi.org/10.1080/00986445.2017.1384921.
113
(252) Hermida, É. B.; Mega, V. I. Transcrystallization Kinetics at the Poly(3-Hydroxybutyrate-
Co-3-Hydroxyvalerate)/Hemp Fibre Interface. Compos. Part A Appl. Sci. Manuf. 2007, 38
(5), 1387–1394. https://doi.org/10.1016/j.compositesa.2006.10.006.
(253) Gatenholm, P.; Kubát, J.; Mathiasson, A. Biodegradable Natural Composites. I. Processing
and Properties. J. Appl. Polym. Sci. 1992, 45 (9), 1667–1677.
https://doi.org/10.1002/app.1992.070450918.
(254) Seggiani, M.; Cinelli, P.; Balestri, E.; Mallegni, N.; Stefanelli, E.; Rossi, A.; Lardicci, C.;
Lazzeri, A. Novel Sustainable Composites Based on Poly(Hydroxybutyrate-Co-
Hydroxyvalerate) and Seagrass Beach-CAST Fibers: Performance and Degradability in
Marine Environments. Materials (Basel). 2018, 11 (5).
https://doi.org/10.3390/ma11050772.
(255) Avella, M.; Martuscelli, E.; Pacucci, B.; Raimo, M.; Marzetti, A. A New Class of
Biodegradable Materials: Poly-3-Hydroxy- Butyrate/ Steam Exploded Straw Fiber
Composites. 1. Thermal and Impact Behaviour. J. Appl. Polym. Sci. 1993, 49, 2091–2103.
(256) Barkoula, N. M.; Garkhail, S. K.; Peijs, T. Biodegradable Composites Based on
Flax/Polyhydroxybutyrate and Its Copolymer with Hydroxyvalerate. Ind. Crops Prod.
2010, 31 (1), 34–42. https://doi.org/10.1016/j.indcrop.2009.08.005.
(257) Muthuraj, R.; Misra, M.; Mohanty, A. K. Reactive Compatibilization and Performance
Evaluation of Miscanthus Biofiber Reinforced Poly(Hydroxybutyrate-Co-
Hydroxyvalerate) Biocomposites. J. Appl. Polym. Sci. 2017, 134 (21), 1–10.
https://doi.org/10.1002/app.44860.
(258) Karnani, R.; Krishnan, M.; Narayan, R. Biofiber-Reinforced Polypropylene Composites.
Polym. Eng. Sci. 1997, 37 (2), 476–483. https://doi.org/10.1002/pen.11691.
(259) Bledzki, A. K.; Mamun, A. A.; Faruk, O. Abaca Fibre Reinforced PP Composites and
Comparison with Jute and Flax Fibre PP Composites. Express Polym. Lett. 2007, 1 (11),
755–762. https://doi.org/10.3144/expresspolymlett.2007.104.
114
(260) Hwang, S. W.; Shim, J. K.; Selke, S.; Soto-Valdez, H.; Rubino, M.; Auras, R. Effect of
Maleic-Anhydride Grafting on the Physical and Mechanical Properties of Poly(L-Lactic
Acid)/Starch Blends. Macromol. Mater. Eng. 2013, 298 (6), 624–633.
https://doi.org/10.1002/mame.201200111.
(261) Avella, M.; Bogoeva-Gaceva, G.; Buzõarovska, A.; Emanuela Errico, M.; Gentile, G.;
Grozdanov, A. Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate)-Based Biocomposite s
Reinforced with Kenaf Fibers. J. Appl. Polym. Sci. 2007, 104 (5), 3192–3200.
https://doi.org/10.1002/app.26057.
(262) Wu, C. S.; Liao, H. T. The Mechanical Properties, Biocompatibility and Biodegradabil ity
of Chestnut Shell Fibre and Polyhydroxyalkanoate Composites. Polym. Degrad. Stab. 2014,
99 (1), 274–282. https://doi.org/10.1016/j.polymdegradstab.2013.10.019.
(263) Lee, H. S.; Cho, D.; Han, S. O. Effect of Natural Fiber Surface Treatments on the Interfacia l
and Mechanical Properties of Henequen/Polypropylene Biocomposites. Macromol. Res.
2008, 16 (5), 411–417. https://doi.org/10.1007/BF03218538.
(264) Lee, S. G.; Choi, S.-S.; Park, W. H.; Cho, D. Characterization of Surface Modified Flax
Fibers and Their Biocomposites with PHB. Macromol. Symp. 2003, 197 (1), 089–100.
https://doi.org/10.1002/masy.200350709.
(265) Zarrinbakhsh, N.; Mohanty, A. K.; Misra, M. Fundamental Studies on Water-Washing of
the Corn Ethanol Coproduct (DDGS) and Its Characterization for Biocomposite
Applications. Biomass and Bioenergy 2013, 55, 251–259.
https://doi.org/10.1016/j.biombioe.2013.02.016.
(266) Mwaikambo L Y; Ansell Martin P. The Effect of Chemical Treatment on the Properties of
Hemp, Sisal, Jute and Kapok for Composite Reinforcement. Die Angew. Makromol. Chemie
1999, 272 (4753), 108–116. https://doi.org/10.1002/(SICI)1522-
9505(19991201)272:1<108::AID-APMC108>3.0.CO;2-9.
(267) Dangtungee, R.; Tengsuthiwat, J.; Boonyasopon, P.; Siengchin, S. Sisal Natural Fiber/Clay-
115
Reinforced Poly(Hydroxybutyrate-Co-Hydroxyvalerate) Hybrid Composites. J.
Thermoplast. Compos. Mater. 2015, 28 (6), 879–895.
https://doi.org/10.1177/0892705714563128.
(268) Kumar Sinha, A.; Narang, H. K.; Bhattacharya, S. Effect of Alkali Treatment on Surface
Morphology of Abaca Fibre. Mater. Today Proc. 2017, 4 (8), 8993–8996.
https://doi.org/10.1016/j.matpr.2017.07.251.
(269) Xie, Y.; Hill, C. A. S.; Xiao, Z.; Militz, H.; Mai, C. Silane Coupling Agents Used for Natural
Fiber/Polymer Composites: A Review. Compos. Part A Appl. Sci. Manuf. 2010, 41 (7), 806–
819. https://doi.org/10.1016/j.compositesa.2010.03.005.
(270) Yew, S. P.; Tang, H. Y.; Sudesh, K. Photocatalytic Activity and Biodegradation of
Polyhydroxybutyrate Films Containing Titanium Dioxide. Polym. Degrad. Stab. 2006, 91
(8), 1800–1807. https://doi.org/10.1016/j.polymdegradstab.2005.11.011.
(271) Montagna, L. S.; Oyama, I. C.; Lamparelli, R. de C. B. C.; Silva, A. P.; Montanheiro, T. L.
D. A.; Lemes, A. P. Evaluation of Biodegradation in Aqueous Medium of
Poly(Hydroxybutyrate-Co-Hydroxyvalerate)/Carbon Nanotubes Films in Respirometr ic
System. J. Renew. Mater. 2019, 7 (2), 117–128. https://doi.org/10.32604/jrm.2019.00036.
(272) Puglia, D.; Fortunati, E.; D’Amico, D. A.; Manfredi, L. B.; Cyras, V. P.; Kenny, J. M.
Influence of Organically Modified Clays on the Properties and Disintegrability in Compost
of Solution Cast Poly(3-Hydroxybutyrate) Films. Polym. Degrad. Stab. 2014, 99 (1), 127–
135. https://doi.org/10.1016/j.polymdegradstab.2013.11.013.
(273) Bhatt, R.; Shah, D.; Patel, K. C.; Trivedi, U. PHA-Rubber Blends: Synthesis,
Characterization and Biodegradation. Bioresour. Technol. 2008, 99 (11), 4615–4620.
https://doi.org/10.1016/j.biortech.2007.06.054.
(274) Kuntanoo, K.; Promkotra, S.; Kaewkannetra, P.; Material, a. Biodegradation of
Polyhydroxybutyrate-Co- Hydroxyvalerate ( PHBV ) Blended with Natural Rubber in Soil
Environment. World Acad. Sci. Eng. Technol. 2013, 7 (12), 1799–1803.
116
(275) Rose, K.; Steinbuchel, A. Biodegradation of Natural Rubber and Related Compounds:
Recent Insights into a Hardly Understood Catabolic Capability of Microorganisms. Appl.
Environ. Microbiol. 2005, 71 (6), 2803–2812. https://doi.org/10.1128/AEM.71.6.2803-
2812.2005.
(276) Le Duigou, A.; Bourmaud, A.; Baley, C. In-Situ Evaluation of Flax Fibre Degradation
during Water Ageing. Ind. Crops Prod. 2015, 70, 190–200.
https://doi.org/10.1016/j.indcrop.2015.03.049.
(277) Freier, T.; Kunze, C.; Nischan, C.; Kramer, S.; Sternberg, K.; Saß, M.; Hopt, U. T.; Schmitz,
K. P. In Vitro and in Vivo Degradation Studies for Development of a Biodegradable Patch
Based on Poly(3-Hydroxybutyrate). Biomaterials 2002, 23 (13), 2649–2657.
https://doi.org/10.1016/S0142-9612(01)00405-7.
(278) Ohura, T.; Aoyagi, Y.; Takagi, K. I.; Yoshida, Y.; Kasuya, K. I.; Doi, Y. Biodegradation of
Poly(3-Hydroxyalkanoic Acids) Fibers and Isolation of Poly(3-Hydroxybutyric Acid)-
Degrading Microorganisms under Aquatic Environments. Polym. Degrad. Stab. 1999, 63
(1), 23–29. https://doi.org/10.1016/S0141-3910(98)00057-3.
(279) Mohanty, A. K.; Khan, M. A.; Hinrichsen, G. Surface Modification of Jute and Its Influence
on Performance of Biodegradable Jute-Fabric/Biopol Composites. Compos. Sci. Technol.
2000, 60 (7), 1115–1124. https://doi.org/10.1016/S0266-3538(00)00012-9.
(280) Bayerl, T.; Geith, M.; Somashekar, A. A.; Bhattacharyya, D. Influence of Fibre Architecture
on the Biodegradability of FLAX/PLA Composites. Int. Biodeterior. Biodegrad. 2014, 96,
18–25. https://doi.org/10.1016/j.ibiod.2014.08.005.
(281) Faibunchan, P.; Nakaramontri, Y.; Chueangchayaphan, W.; Pichaiyut, S.; Kummerlöwe,
C.; Vennemann, N.; Nakason, C. Novel Biodegradable Thermoplastic Elastomer Based on
Poly(Butylene Succinate) and Epoxidized Natural Rubber Simple Blends. J. Polym.
Environ. 2018, 26 (7), 2867–2880. https://doi.org/10.1007/s10924-017-1173-4.
(282) Kirk, T. K.; Farrell, R. L. Enzymatic “Combustion”: The Microbial Degradation of Lignin.
117
Annu. Rev. Microbiol. 1987, 41 (1), 465–501.
https://doi.org/10.1146/annurev.mi.41.100187.002341.
(283) Phithakrotchanakoon, C.; Rudeekit, Y.; Tanapongpipat, S.; Leejakpai, T.; Aiba, S. I.; Noda,
I.; Champreda, V. Microbial Degradation and Physico-Chemical Alteration of
Polyhydroxyalkanoates by a Thermophilic Streptomyces Sp. Biologia (Bratisl). 2009, 64
(2), 246–251. https://doi.org/10.2478/s11756-009-0050-6.
(284) Wu, C. S. Mechanical Properties, Biocompatibility, and Biodegradation of Cross-Linked
Cellulose Acetate-Reinforced Polyester Composites. Carbohydr. Polym. 2014, 105 (1), 41–
48. https://doi.org/10.1016/j.carbpol.2014.01.062.
(285) Gardner, R. M.; Buchanan, C. M.; Komarek, R.; Dorschel, D.; Boggs, C.; White, A. W.
Compostability of Cellulose Acetate Films. J. Appl. Polym. Sci. 1994, 52 (10), 1477–1488.
https://doi.org/10.1002/app.1994.070521012.
(286) El-Shafee, E.; Saad, G. R.; Fahmy, S. M. Miscibility, Crystallization and Phase Structure
of Poly(3-Hydroxybutyrate)/Cellulose Acetate Butyrate Blends. Eur. Polym. J. 2001, 37
(10), 2091–2104. https://doi.org/10.1016/S0014-3057(01)00097-0.
(287) Mousavioun, P.; George, G. A.; Doherty, W. O. S. Environmental Degradation of
Lignin/Poly(Hydroxybutyrate) Blends. Polym. Degrad. Stab. 2012, 97 (7), 1114–1122.
https://doi.org/10.1016/j.polymdegradstab.2012.04.004.
(288) Kratsch, H. A.; Schrader, J. A.; McCabe, K. G.; Srinivasan, G.; Grewell, D.; Graves, W. R.
Performance and Biodegradation in Soil of Novel Horticulture Containers Made from
Bioplastics and Biocomposites. Horttechnology 2015, 25 (1), 119–131.
https://doi.org/10.21273/horttech.25.1.119.
(289) Wei, L.; Liang, S.; McDonald, A. G. Thermophysical Properties and Biodegradation
Behavior of Green Composites Made from Polyhydroxybutyrate and Potato Peel Waste
Fermentation Residue. Ind. Crops Prod. 2015, 69, 91–103.
https://doi.org/10.1016/j.indcrop.2015.02.011.
118
(290) Mahmood, A. U.; Greenman, J.; Scragg, A. H. Orange and Potato Peel Extracts: Analysis
and Use as Bacillus Substrates for the Production of Extracellular Enzymes in Continuous
Culture. Enzyme Microb. Technol. 1998, 22, 130–137.
(291) Peterson, S.; Jayaraman, K.; Bhattacharyya, D. Forming Performance and Biodegradabil ity
of Woodfibre-BiopolTM Composites. Compos. Part A Appl. Sci. Manuf. 2002, 33 (8), 1123–
1134. https://doi.org/10.1016/S1359-835X(02)00046-5.
(292) Carvalheiro, F.; Silva-Fernandes, T.; Duarte, L. C.; Gírio, F. M. Wheat Straw
Autohydrolysis: Process Optimization and Products Characterization. Appl. Biochem.
Biotechnol. 2009, 153 (1–3), 84–93. https://doi.org/10.1007/s12010-008-8448-0.
(293) Clemons, C. M. Wood Flour. In Functional Fillers for Plastics; Xanthos, M., Ed.; Wiley-
VCH Verlag GmbH & Co. KGaA: Weinheim, FRG, 2010; pp 249–270.
https://doi.org/10.1002/3527605096.ch15.
(294) Ismail, M. S.; Waliuddin, A. M. Effect of Rice Husk Ash on High Strength Concrete.
Constr. Build. Mater. 1996, 10 (7), 521–526. https://doi.org/10.1016/0950-0618(96)00010-
4.
(295) Bolanho, B. C.; Danesi, E. D. G.; Beléia, A. D. P. Carbohydrate Composition of Peach Palm
(Bactris Gasipaes Kunth) by-Products Flours. Carbohydr. Polym. 2015, 124, 196–200.
https://doi.org/10.1016/j.carbpol.2015.02.021.
(296) Saragih, S. W.; Lubis, R.; Wirjosentono, B.; Eddyanto. Characteristic of Abaca (Musa
Textilis) Fiber from Aceh Timur as Bioplastic. AIP Conf. Proc. 2018, 2049 (December).
https://doi.org/10.1063/1.5082463.
(297) Liu, K. Chemical Composition of Distillers Grains, a Review. J. Agric. Food Chem. 2011,
59 (5), 1508–1526. https://doi.org/10.1021/jf103512z.
(298) Øverland, M.; Mydland, L. T.; Skrede, A. Marine Macroalgae as Sources of Protein and
Bioactive Compounds in Feed for Monogastric Animals. J. Sci. Food Agric. 2019, 99 (1),
119
13–24. https://doi.org/10.1002/jsfa.9143.
(299) Ismojo; Yuanita, E.; Rosa, E. M.; Calvin, L.; Chalid, M. Effect of Time Alkali Treatment
on Chemical Composition and Tensile Strength Properties of Kenaf Single Fibers. Proc.
5Th Int. Symp. Appl. Chem. 2019 2019, 2175 (November), 020059.
https://doi.org/10.1063/1.5134623.
(300) Abdul Khalil, H. P. S.; Ismail, H. Effect of Acetylation and Coupling Agent Treatments
upon Biological Degradation of Plant Fibre Reinforced Polyester Composites. Polym. Test.
2000, 20 (1), 65–75. https://doi.org/10.1016/S0142-9418(99)00080-X.
(301) Calabia, B. P.; Ninomiya, F.; Yagi, H.; Oishi, A.; Taguchi, K.; Kunioka, M.; Funabashi, M.
Biodegradable Poly(Butylene Succinate) Composites Reinforced by Cotton Fiber with
Silane Coupling Agent. Polymers (Basel). 2013, 5 (1), 128–141.
https://doi.org/10.3390/polym5010128.
(302) Thomas, S.; Shumilova, A. A.; Kiselev, E. G.; Baranovsky, S. V.; Vasiliev, A. D.; Nemtsev,
I. V.; Kuzmin, A. P.; Sukovatyi, A. G.; Avinash, R. P.; Volova, T. G. Thermal, Mechanica l
and Biodegradation Studies of Biofiller Based Poly-3-Hydroxybutyrate Biocomposites. Int.
J. Biol. Macromol. 2019, No. xxxx. https://doi.org/10.1016/j.ijbiomac.2019.11.112.
(303) Harmaen, A. S.; Khalina, A.; Ali, H. M.; Azowa, I. N. Thermal, Morphological, and
Biodegradability Properties of Bioplastic Fertilizer Composites Made of Oil Palm Biomass,
Fertilizer, and Poly(Hydroxybutyrate-Co-Valerate). Int. J. Polym. Sci. 2016, 2016.
https://doi.org/10.1155/2016/3230109.
(304) Sanchez-Safont, E. L.; Gonzalez-Ausejo, J.; Gamez-Perez, J.; Lagaron, J. M.; Cabedo, L.
Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate)/ Purified Cellulose Fiber Composites by
Melt Blending: Characterization and Degradation in Composting Conditions. J. Renew.
Mater. 2016, 4 (2), 123–132. https://doi.org/10.7569/JRM.2015.634127.
(305) Arrieta, M. P.; Fortunati, E.; Dominici, F.; Rayón, E.; López, J.; Kenny, J. M. PLA-
PHB/Cellulose Based Films: Mechanical, Barrier and Disintegration Properties. Polym.
120
Degrad. Stab. 2014, 107, 139–149.
https://doi.org/10.1016/j.polymdegradstab.2014.05.010.
(306) Imam, S. H.; Chen, L.; Gorden, S. H.; Shogren, R. L.; Weisleder, D.; Greene, R. V.
Biodegradation of Injection Molded Starch-Poly(3-Hydroxybutyrate-Co-3-
Hydroxyvalerate) Blends in a Natural Compost Environment. J. Environ. Polym. Degrad.
1998, 6 (2), 91–98. https://doi.org/10.1023/A:1022806222158.
(307) Schrader, J. A.; McCabe, K. G.; Grewell, D.; Graves, W. R. Bioplastics and Biocomposite s
for Sustainable Horticultural Containers: Performance and Biodegradation in Home
Compost. Acta Hortic. 2017, 1170, 1101–1108.
https://doi.org/10.17660/ActaHortic.2017.1170.142.
(308) Muniyasamy, S.; Reddy, M. M.; Misra, M.; Mohanty, A. Biodegradable Green Composites
from Bioethanol Co-Product and Poly(Butylene Adipate-Co-Terephthalate). Ind. Crops
Prod. 2013, 43 (1), 812–819. https://doi.org/10.1016/j.indcrop.2012.08.031.
(309) Ismail, A. M.; Gamal, M. A. B. Water Resistance, Mechanical Properties, and
Biodegradability of Poly(3-Hydroxybutyrate)/Starch Composites. J. Appl. Polym. Sci.
2010, 115 (5), 2813–2819. https://doi.org/10.1002/app.31181.
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-
acrylic-co-glycidyl methacrylate). To increase the ductility and impact properties of PHBV, and
to investigate a new biodegradable PHBV-based blend for sustainable packaging, CA was
compatibilized with TEC. PHBV and plasticized CA (pCA) blends showed complete immiscibility
through separate glass transition and melting peak temperatures in DSC; despite the similar Hansen
solubility parameters of PHBV, CA and TEC indicating partial miscibility. Phase separation
between PHBV and pCA was clearly observed by scanning electron microscopy (SEM).
PHBV/pCA (70/30) blends had improved impact strength, exceeding neat PHBV and pCA, which
is attributed to PHBV porosity induced by degradation from the high processing temperature.
During processing, the plasticizer migrated from CA to PHBV and partially plasticized it, as
evidenced through DSC analysis. The melt temperature of PHBV was reduced, which was
confirmed by double melting peaks, representing the formation of secondary crystallites at a lower
temperature. Due to processing at high temperature (210-220 °C), significant porosity was
observed in PHBV/pCA (30/70) blend in SEM analysis. Consequently, the impact strength was
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.
3.5 References
(1) Giacovelli, C.; Zamparo, A.; Wehrli, A.; Alverson, K. Single-Use Plastic: A Roadmap for
Sustainability; 2018. https://doi.org/DOI: 10.1016/0145-305X(89)90168-7.
(2) Stevens, E. S. What Makes Green Plastics Green? Biocycle 2003, 44 (3), 24–27.
(3) Kasuya, K.; Takagi, K.; Ishiwatari, S.; Yoshida, Y.; Doi, Y. Biodegradabilities of Various
Aliphatic Polyesters in Natural Waters. Polym. Degrad. Stab. 2002, 59 (1–3), 327–332.
https://doi.org/10.1016/s0141-3910(97)00155-9.
(4) Shibata, M.; Oyamada, S.; Kobayashi, S.; Yaginuma, D. Mechanical Properties and
Biodegradability of Green Composites Based on Biodegradable Polyesters and Lyocell
Fabric. J. Appl. Polym. Sci. 2004, 92 (6), 3857–3863. https://doi.org/10.1002/app.20405.
(5) Handbook of Biodegradable Polymers; Bastioli, C., Ed.; Rapra Technology Limited, 2005.
146
(6) Sheu, D. S.; Chen, W. M.; Yang, J. Y.; Chang, R. C. Thermophilic Bacterium Caldimonas
Taiwanensis Produces Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) from Starch and
Valerate as Carbon Sources. Enzyme Microb. Technol. 2009, 44 (5), 289–294.
https://doi.org/10.1016/j.enzmictec.2009.01.004.
(7) Ramkumar, D. H. S.; Bhattacharya, M. Steady Shear and Dynamic Properties of
Biodegradable Polyesters. Polym. Eng. Sci. 1998, 38 (9), 1426–1435.
(8) Corre, Y. M.; Bruzaud, S.; Audic, J.-L.; Grohens, Y. Morphology and Functional Properties
of Commercial Polyhydroxyalkanoates: A Comprehensive and Comparative Study. Polym.
Test. 2012, 31 (2), 226–235. https://doi.org/10.1016/j.polymertesting.2011.11.002.
(9) Bledzki, A. K.; Jaszkiewicz, A. Mechanical Performance of Biocomposites Based on PLA
and PHBV Reinforced with Natural Fibres - A Comparative Study to PP. Compos. Sci.
Technol. 2010, 70 (12), 1687–1696. https://doi.org/10.1016/j.compscitech.2010.06.005.
(10) Shishatskaya, E. I. Biomedical Investigations of Biodegradable PHAs. Macromol. Symp.
2008, 269 (1), 65–81. https://doi.org/10.1002/masy.200850909.
(11) Lagarón, J. M.; López-Rubio, A.; José Fabra, M. Bio-Based Packaging. J. Appl. Polym. Sci.
2016, 133 (2). https://doi.org/10.1002/app.42971.
(12) Kim, C. H.; Choi, E. J.; Park, J. K. Effect of PEG Molecular Weight on the Tensile
Toughness of Starch/PCL/PEG Blends. J. Appl. Polym. Sci. 2000, 77 (9), 2049–2056.
https://doi.org/10.1002/1097-4628(20000829)77:9<2049::AID-APP22>3.0.CO;2-C.
(13) Chandra, R.; Rustgi, R. BIODEGRADABLE POLYMERS. Prog. Polym. Sci. 1998, 23
(97), 1273–1335. https://doi.org/10.1016/S0079-6700(97)00039-7.
(14) Vroman, I.; Tighzert, L. Biodegradable Polymers. Materials (Basel). 2009, 2 (2), 307–344.
https://doi.org/10.3390/ma2020307.
(15) Kumar, A.; Sinha-Ray, S. A Review on Biopolymer-Based Fibers via Electrospinning and
Solution Blowing and Their Applications. Fibers 2018, 6 (3), 45.
https://doi.org/10.3390/fib6030045.
(16) Robeson, L. M. Fundamentals of Polymer Blends. In Polymer Blends A Comprehensive
Review; Carl Hanser Verlag GmbH & Co. KG, 2007; pp 11–64.
https://doi.org/10.3139/9783446436503.002.
(17) Barlow, J. W.; Paul, D. R. Mechanical Compatibilization of Immiscible Blends. Polym.
147
Eng. Sci. 1984, 24 (8), 525–534. https://doi.org/10.1002/pen.760240804.
(18) Muhamad, I. I.; Joon, L. K.; Noor, M. A. M. Comparing the Degradation of Poly-β-
(Hydroxybutyrate), Poly-β –(Hydroxybutyrate-Co-Valerate)(PHBV) and PHBV / Cellulo se
Triacetate Blend. Malaysian Polym. J. 2006, 1 (1), 39–46.
(19) Pizzoli, M.; Scandola, M.; Ceccorulli, G. Crystallization Kinetics and Morphology of
Poly(3-Hydroxybutyrate)/Cellulose Ester Blends. Macromolecules 1994, 27 (17), 4755–
4761. https://doi.org/10.1021/ma00095a016.
(20) Buchanan, C. M.; Gedon, S. C.; White, A. W.; Wood, M. D. Cellulose Acetate Butyrate and
Poly(Hydroxybutyrate-Co-Valerate) Copolymer Blends. Macromolecules 1992, 25 (26),
7373–7381. https://doi.org/10.1021/ma00052a046.
(21) Lotti, N.; Scandola, M. Miscibility of Bacterial Poly(3-Hydroxybutyrate-Co-3-
Hydroxyvalerate) with Ester Substituted Celluloses. Polym. Bull. 1992, 29 (3–4), 407–413.
https://doi.org/10.1007/BF00944838.
(22) Liao, Q.; Tsui, A.; Billington, S.; Frank, C. W. Extruded Foams from Microbial Poly(3-
Hydroxybutyrate-Co-3-Hydroxyvalerate) and Its Blends with Cellulose Acetate Butyrate.
Polym. Eng. Sci. 2012, 52 (7), 1495–1508. https://doi.org/10.1002/pen.23087.
(23) Gardner, R. M.; Buchanan, C. M.; Komarek, R.; Dorschel, D.; Boggs, C.; White, A. W.
Compostability of Cellulose Acetate Films. J. Appl. Polym. Sci. 1994, 52 (10), 1477–1488.
https://doi.org/10.1002/app.1994.070521012.
(24) Wojciechowska, P.; Heimowska, A.; Foltynowicz, Z.; Rutkowska, M. Degradability of
Organic-Inorganic Cellulose Acetate Butyrate Hybrids in Sea Water. Polish J. Chem.
Technol. 2011, 13 (2), 29–34. https://doi.org/10.2478/v10026-011-0020-y.
(25) Mohanty, A. K.; Wibowo, A.; Misra, M.; Drzal, L. T. Development of Renewable
Resource-Based Cellulose Acetate Bioplastic: Effect of Process Engineering on the
Performance of Cellulosic Plastics. Polym. Eng. Sci. 2003, 43 (5), 1151–1161.
https://doi.org/10.1002/pen.10097.
(26) Wibowo, A. C.; Misra, M.; Park, H. M.; Drzal, L. T.; Schalek, R.; Mohanty, A. K.
Biodegradable Nanocomposites from Cellulose Acetate: Mechanical, Morphological, and
Thermal Properties. Compos. Part A Appl. Sci. Manuf. 2006, 37 (9), 1428–1433.
https://doi.org/10.1016/j.compositesa.2005.06.019.
148
(27) Choi, J. S.; Park, W. H. Effect of Biodegradable Plasticizers on Thermal and Mechanica l
Properties of Poly(3-Hydroxybutyrate). Polym. Test. 2004, 23 (4), 455–460.
https://doi.org/10.1016/j.polymertesting.2003.09.005.
(28) Park, H. M.; Liang, X.; Mohanty, A. K.; Misra, M.; Drzal, L. T. Effect of Compatibilize r
on Nanostructure of the Biodegradable Cellulose Acetate/Organoclay Nanocomposites.
Macromolecules 2004, 37 (24), 9076–9082. https://doi.org/10.1021/ma048958s.
(29) Souza, D.; Castillo, T. E.; Rodríguez, R. J. S. Effects of Hydroxyvalerate Contents in
Thermal Degradation Kinetic of Cellulose Acetate Propionate/Poly(3-Hydroxyalkanoates )
Blends. J. Therm. Anal. Calorim. 2012, 109 (3), 1353–1364.
https://doi.org/10.1007/s10973-011-2152-y.
(30) El-Shafee, E.; Saad, G. R.; Fahmy, S. M. Miscibility, Crystallization and Phase Structure
of Poly(3-Hydroxybutyrate)/Cellulose Acetate Butyrate Blends. Eur. Polym. J. 2001, 37
(10), 2091–2104. https://doi.org/10.1016/S0014-3057(01)00097-0.
(31) Duangphet, S.; Szegda, D.; Song, J.; Tarverdi, K. The Effect of Chain Extender on Poly(3-
Hydroxybutyrate-Co-3-Hydroxyvalerate): Thermal Degradation, Crystallization, and
Rheological Behaviours. J. Polym. Environ. 2014, 22 (1), 1–8.
https://doi.org/10.1007/s10924-012-0568-5.
(32) Srithep, Y.; Sabo, R.; Clemons, C.; Turng, L.; Pilla, S.; Peng, J. Effect of Material
Parameters on Mechanical Properties of Biodegradable Polymers / Nanofibrilla ted
Cellulose ( Nfc ) Nanocomposites. 2012, 4–8.
(33) Mathieu, D. Pencil and Paper Estimation of Hansen Solubility Parameters. ACS Omega
2018, 3 (12), 17049–17056. https://doi.org/10.1021/acsomega.8b02601.
(34) Snowdon, M. R.; Mohanty, A. K.; Misra, M. Miscibility and Performance Evaluation of
Biocomposites Made from Polypropylene/Poly(Lactic Acid)/Poly(Hydroxybutyrate- Co -
Hydroxyvalerate) with a Sustainable Biocarbon Filler. Am. Chem. Soc. 2017, 2 (10), 6446–
6454. https://doi.org/10.1021/acsomega.7b00983.
(35) Puleo, A. C.; Paul, D. R. The Effect of Degree of Acetylation on Gas Sorption and Transport
Behanviour in Cellulose Acetate. J. Memb. Sci. 1989, 47, 301–332.
(36) Van Krevelen, D. W.; Te Nijenhuis, K. Cohesive Properties and Solubility. In Properties of
Polymers; 2009; pp 189–227. https://doi.org/10.1016/B978-0-08-054819-7.00007-8.
149
(37) Forster, A.; Hempenstall, J.; Tucker, I.; Rades, T. Selection of Excipients for Melt Extrus ion
with Two Poorly Water-Soluble Drugs by Solubility Parameter Calculation and Thermal
Analysis. Int. J. Pharm. 2001, 226 (1–2), 147–161. https://doi.org/10.1016/S0378-
5173(01)00801-8.
(38) Wang, B.; Chen, J.; Peng, H.; Gai, J.; Kang, J.; Cao, Y. Investigation on Changes in the
Miscibility, Morphology, Rheology and Mechanical Behavior of Melt Processed Cellulo se
Acetate through Adding Polyethylene Glycol as a Plasticizer. J. Macromol. Sci. Part B
Phys. 2016, 55 (9), 894–907. https://doi.org/10.1080/00222348.2016.1217185.
(39) Yasuniwa, M.; Tsubakihara, S.; Sugimoto, Y.; Nakafuku, C. Thermal Analysis of the
Double-Melting Behavior of Poly(L-Lactic Acid). J. Polym. Sci. Part B Polym. Phys. 2004,
42 (1), 25–32. https://doi.org/10.1002/polb.10674.
(40) Baldenegro-Perez, L. A.; Navarro-Rodriguez, D.; Medellin-Rodriguez, F. J.; Hsiao, B.;
Avila-Orta, C. A.; Sics, I. Molecular Weight and Crystallization Temperature Effects on
Poly(Ethylene Terephthalate) (PET) Homopolymers, an Isothermal Crystallizat ion
Analysis. Polymers (Basel). 2014, 6 (2), 583–600. https://doi.org/10.3390/polym6020583.
(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.
https://doi.org/10.1007/s10924-017-1128-9.
(42) Bonartsev, A. P.; Boskhomodgiev, A. P.; Iordanskii, A. L.; Bonartseva, G. A.; Rebrov, A.
V.; Makhina, T. K.; Myshkina, V. L.; Yakovlev, S. A.; Filatova, E. A.; Ivanov, E. A.; et al.
Hydrolytic Degradation of Poly(3-Hydroxybutyrate), Polylactide and Their Derivatives :
Kinetics, Crystallinity, and Surface Morphology. Mol. Cryst. Liq. Cryst. 2012, 556, 288–
300. https://doi.org/10.1080/15421406.2012.635982.
(43) Chen, X.; Hou, G.; Chen, Y.; Yang, K.; Dong, Y.; Zhou, H. Effect of Molecular Weight on
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.
(44) Enriquez, E.; Mohanty, A. K.; Misra, M. Biobased Blends of Poly(Propylene Carbonate)
and Poly(Hydroxybutyrate-Co-Hydroxyvalerate): Fabrication and Characterization. J.
Appl. Polym. Sci. 2017, 134 (5). https://doi.org/10.1002/app.44420.
150
(45) Zhu, Y.; Mehta, K. A.; Mcginity, J. W.; Zhu, Y.; Mehta, K. A.; Mcginity, J. W. Influence
of Plasticizer Level on the Drug Release from Sustained Release Film Coated and Hot-Melt
Extruded Dosage Forms Influence of Plasticizer Level on the Drug Release from Sustained
Release Film Coated and Hot-Melt Extruded Dosage Forms. Pharm. Dev. Technol. 2008,
11 (3), 285–294. https://doi.org/10.1080/10409230600767551.
(46) Ferfera-Harrar, H.; Dairi, N. Green Nanocomposite Films Based on Cellulose Acetate and
Biopolymer-Modified Nanoclays: Studies on Morphology and Properties. Iran. Polym. J.
(English Ed. 2014, 23 (12), 917–931. https://doi.org/10.1007/s13726-014-0286-z.
(47) Shahsavarani, A. A. Improvement of the Properties of Novel Bioplastics Through Reactive
Compatibilization, Brunel University, 2016.
(48) Gerard, T.; Budtova, T.; Podshivalov, A.; Bronnikov, S. Polylactide/Poly(Hydroxybutyrate -
Co-Hydroxyvalerate) Blends: Morphology and Mechanical Properties. Express Polym. Lett.
2014, 8 (8), 609–617. https://doi.org/10.3144/expresspolymlett.2014.64.
(49) Mohanty, A. K.; Wibowo, A.; Misra, M.; Drzal, L. T. Effect of Process Engineering on the
Performance of Natural Fiber Reinforced Cellulose Acetate Biocomposites. Compos. Part
A Appl. Sci. Manuf. 2004, 35 (3), 363–370.
https://doi.org/10.1016/j.compositesa.2003.09.015.
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 Materials and Methods
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 Thermal Properties
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
𝑋𝐶 = (∆𝐻𝑚
∆𝐻𝑚0 × 𝑤𝑓
) × 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.
4.2.5 Mechanical Properties
4.2.5.1 Tensile and Flexural Properties
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.
4.2.6 Scanning Electron Microscopy (SEM)
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
4.3 Results and Discussion
4.3.1 Mechanical Properties
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
lus
(GP
a)
A)
0
20
40
60
Ten
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
4.3.7 Marine Biodegradation
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
vo
luti
on
(m
g)
Time (day)
A)
0 50 100 150 200 250 300 350 400 450 500
0
10
20
30
40
50
60
70
80
90
100
110B)
% B
iod
eg
ra
da
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.
4.5 References
(1) IfBB- Institute for Bioplastics And Biocomposites. Biopolymers Facts and Statistics 2015;
2015.
(2) Geyer, R.; Jambeck, J. R.; Law, K. L. Production, Use, and Fate of All Plastics Ever Made. Sci. Adv. 2017, 3 (7), 25–29. https://doi.org/10.1126/sciadv.1700782.
(3) Nova Institute. European Bioplastics https://www.european-bioplastics.org/ (accessed Mar 11, 2020).
(4) Storz, H.; Vorlop, K.-D. Bio-Based Plastics: Status, Challenges and Trends. Landbauforsch. Volkenrode 2014, 63 (4), 321–332. https://doi.org/10.3220/LBF_2013_321-332.
(5) New Plastic Economy. The New Plastic Economy 2019 Progress Report; 2019.
(6) Jambeck, J.; Geyer, R.; Wilcox, C.; Siegler, T. R.; Perryman, M.; Andrady, A.; Narayan, R.; Law, K. L. Plastic Waste Inputs from Land into the Ocean. 2015, 347 (6223). https://doi.org/10.1126/science.1260352.
(7) Verhoogt, H.; Ramsay, B. A.; Favis, B. D. Polymer Blends Containing Poly(3-
Hydroxyalkanoate)S. Polymer (Guildf). 1994, 35 (24), 5155–5169. https://doi.org/10.1016/0032-3861(94)90465-0.
(8) Vroman, I.; Tighzert, L. Biodegradable Polymers. Materials (Basel). 2009, 2 (2), 307–344. https://doi.org/10.3390/ma2020307.
(9) Giacovelli, C.; Zamparo, A.; Wehrli, A.; Alverson, K. Single-Use Plastic: A Roadmap for Sustainability; 2018. https://doi.org/DOI: 10.1016/0145-305X(89)90168-7.
(10) Gironi, F.; Piemonte, V. Bioplastics and Petroleum-Based Plastics: Strengths and Weaknesses. Energy Sources, Part A Recover. Util. Environ. Eff. 2011, 33 (21), 1949–1959.
https://doi.org/10.1080/15567030903436830.
(11) van den Oever, M.; Molenveld, K.; van der Zee, M.; Bos, H. Bio-Based and Biodegradable
169
Plastics : Facts and Figures : Focus on Food Packaging in the Netherlands; 2017. https://doi.org/10.18174/408350.
(12) Puls, J.; Wilson, S. A.; Hölter, D. Degradation of Cellulose Acetate-Based Materials: A
Review. J. Polym. Environ. 2011, 19 (1), 152–165. https://doi.org/10.1007/s10924-010-0258-0.
(13) Muniyasamy, S.; Ofosu, O.; John, M. J.; Anandjiwala, R. D. Mineralization of Poly(Lactic Acid) (PLA), Poly(3-Hydroxybutyrate-Co-Valerate) (PHBV) and PLA/PHBV Blend in Compost and Soil Environments. J. Renew. Mater. 2016, 4 (2), 133–145.
https://doi.org/10.7569/JRM.2016.634104.
(14) Dilkes-Hoffman, L. S.; Lant, P. A.; Laycock, B.; Pratt, S. The Rate of Biodegradation of PHA Bioplastics in the Marine Environment: A Meta-Study. Mar. Pollut. Bull. 2019, 142 (February), 15–24. https://doi.org/10.1016/j.marpolbul.2019.03.020.
(15) Chen, H. Assessment of Biodegradation in Different Environmental Compartments of
Blends and Composites Based on Microbial Poly ( Hydroxyalkanoate )S, University of Pisa, 2012.
(16) Volova, T. G.; Boyandin, A. N.; Vasiliev, A. D.; Karpov, V. A.; Prudnikova, S. V.; Mishukova, O. V.; Boyarskikh, U. A.; Filipenko, M. L.; Rudnev, V. P.; Bá Xuân, B.; et al. Biodegradation of Polyhydroxyalkanoates (PHAs) in Tropical Coastal Waters and
Identification of PHA-Degrading Bacteria. Polym. Degrad. Stab. 2010, 95 (12), 2350–2359. https://doi.org/10.1016/j.polymdegradstab.2010.08.023.
(17) Yoon, J. S.; Chang, M. C.; Kim, M. N.; Kang, E. J.; Kim, C.; Chin, I. J. Compatibility and Fungal Degradation of Poly[(R)-3-Hydroxybutyrate]/Aliphatic Copolyester Blend. J.
Polym. Sci. Part B Polym. Phys. 1996, 34 (15), 2543–2551. https://doi.org/10.1002/(SICI)1099-0488(19961115)34:15<2543::AID-POLB2>3.0.CO;2-
U.
(18) Kasuya, K.; Takagi, K.; Ishiwatari, S.; Yoshida, Y.; Doi, Y. Biodegradabilities of Various
Aliphatic Polyesters in Natural Waters. Polym. Degrad. Stab. 2002, 59 (1–3), 327–332. https://doi.org/10.1016/s0141-3910(97)00155-9.
(19) Shibata, M.; Oyamada, S.; Kobayashi, S.; Yaginuma, D. Mechanical Properties and Biodegradability of Green Composites Based on Biodegradable Polyesters and Lyocell
Fabric. J. Appl. Polym. Sci. 2004, 92 (6), 3857–3863. https://doi.org/10.1002/app.20405.
(20) Deroiné, M.; César, G.; Le Duigou, A.; Davies, P.; Bruzaud, S. Natural Degradation and
Biodegradation of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) in Liquid and Solid Marine Environments. J. Polym. Environ. 2015, 23 (4), 493–505.
https://doi.org/10.1007/s10924-015-0736-5.
(21) Seggiani, M.; Cinelli, P.; Balestri, E.; Mallegni, N.; Stefanelli, E.; Rossi, A.; Lardicci, C.;
Lazzeri, A. Novel Sustainable Composites Based on Poly(Hydroxybutyrate-Co-
170
Hydroxyvalerate) and Seagrass Beach-CAST Fibers: Performance and Degradability in Marine Environments. Materials (Basel). 2018, 11 (5).
https://doi.org/10.3390/ma11050772.
(22) Imam, S. H.; Gordon, S. H.; Shogren, R. L.; Tosteson, T. R.; Govind, N. S.; Greene, R. V. Degradation of Starch–Poly(b-Hydroxybutyrate-Co-b-Hydroxyvalerate) Bioplastics in Tropical Coastal Waters. Appl. Environ. Microbiol. 1999, 65 (2), 431–437.
(23) Madbouly, S. A.; Schrader, J. A.; Srinivasan, G.; Liu, K.; McCabe, K. G.; Grewell, D.; Graves, W. R.; Kessler, M. R. Biodegradation Behavior of Bacterial-Based
Polyhydroxyalkanoate (PHA) and DDGS Composites. Green Chem. 2014, 16 (4), 1911–1920. https://doi.org/10.1039/c3gc41503a.
(24) Batista, K. C.; Silva, D. A. K.; Coelho, L. A. F.; Pezzin, S. H.; Pezzin, A. P. T. Soil Biodegradation of PHBV/Peach Palm Particles Biocomposites. J. Polym. Environ. 2010, 18
(3), 346–354. https://doi.org/10.1007/s10924-010-0238-4.
(25) Muthuraj, R.; Misra, M.; Mohanty, A. K. Biodegradable Biocomposites from Poly(Butylene Adipate-Co-Terephthalate) and Miscanthus: Preparation, Compatibilization, and Performance Evaluation. J. Appl. Polym. Sci. 2017, 134 (43), 1–9.
https://doi.org/10.1002/app.45448.
(26) Javadi, A.; Srithep, Y.; Lee, J.; Pilla, S.; Clemons, C.; Gong, S.; Turng, L. S. Processing
and Characterization of Solid and Microcellular PHBV/PBAT Blend and Its RWF/Nanoclay Composites. Compos. Part A Appl. Sci. Manuf. 2010, 41 (8), 982–990.
https://doi.org/10.1016/j.compositesa.2010.04.002.
(27) Scandola, M.; Focarete, M. L.; Adamus, G.; Sikorska, W.; Baranowska, I.; Świerczek, S.;
Gnatowski, M.; Kowalczuk, M.; Jedliński, Z. Polymer Blends of Natural Poly(3-Hydroxybutyrate- Co -3-Hydroxyvalerate) and a Synthetic Atactic Poly(3-
Hydroxybutyrate). Characterization and Biodegradation Studies. Macromolecules 1997, 30 (9), 2568–2574. https://doi.org/10.1021/ma961431y.
(28) Enriquez, E.; Mohanty, A. K.; Misra, M. Biobased Blends of Poly(Propylene Carbonate) and Poly(Hydroxybutyrate-Co-Hydroxyvalerate): Fabrication and Characterization. J. Appl. Polym. Sci. 2017, 134 (5), 1–10. https://doi.org/10.1002/app.44420.
(29) Liu, B.; Koc, A. B. Mechanical Properties of Switchgrass and Miscanthus. Trans. ASABE
2017, 60 (3), 581–590. https://doi.org/10.13031/trans.11925.
(30) Muthuraj, R.; Misra, M.; Mohanty, A. K. Reactive Compatibilization and Performance
Evaluation of Miscanthus Biofiber Reinforced Poly(Hydroxybutyrate-Co-Hydroxyvalerate) Biocomposites. J. Appl. Polym. Sci. 2017, 134 (21), 1–10.
https://doi.org/10.1002/app.44860.
(31) Zaidi, S. Z. A.; Crosky, A. High Strength, High Toughness Polyhydroxybutyrate-Co-
Valerate Based Biocomposites. Mater. Metall. Eng. 2017, 11 (1), 93–97.
171
(32) Bledzki, A. K.; Jaszkiewicz, A. Mechanical Performance of Biocomposites Based on PLA and PHBV Reinforced with Natural Fibres - A Comparative Study to PP. Compos. Sci.
Technol. 2010, 70 (12), 1687–1696. https://doi.org/10.1016/j.compscitech.2010.06.005.
(33) Thomason, J. L.; Rudeiros-Fernández, J. L. A Review of the Impact Performance of Natural Fiber Thermoplastic Composites. Front. Mater. 2018, 5 (September), 1–18. https://doi.org/10.3389/fmats.2018.00060.
(34) Naebe, M.; Abolhasani, M. M.; Khayyam, H.; Amini, A.; Fox, B. Crack Damage in Polymers and Composites: A Review. Polym. Rev. 2016, 56 (1), 31–69.
https://doi.org/10.1080/15583724.2015.1078352.
(35) Solle, M. A.; Arroyo, J.; Burgess, M. H.; Warnat, S.; Ryan, C. A. Value-Added Composite Bioproducts Reinforced with Regionally Significant Agricultural Residues. Compos. Part A Appl. Sci. Manuf. 2019, 124 (April), 105441.
https://doi.org/10.1016/j.compositesa.2019.05.009.
(36) Deroiné, M.; Le Duigou, A.; Corre, Y. M.; Le Gac, P. Y.; Davies, P.; César, G.; Bruzaud, S. Seawater Accelerated Ageing of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate ). Polym. Degrad. Stab. 2014, 105 (1), 237–247.
https://doi.org/10.1016/j.polymdegradstab.2014.04.026.
(37) Rodi, E. G.; Langlois, V.; Renard, E.; Sansalone, V.; Lemaire, T. Biocomposites Based on
Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) (PHBHV) and Miscanthus Giganteus Fibers with Improved Fiber/Matrix Interface. Polymers (Basel). 2018, 10 (5).
https://doi.org/10.3390/polym10050509.
(38) Lu, H. Processing and Characterization of Bio-Based Composites, Iowa State University,
2014. https://doi.org/10.31274/etd-180810-2811.
(39) Reis, K. C.; Pereira, J.; Smith, A. C.; Carvalho, C. W. P.; Wellner, N.; Yakimets, I.
Characterization of Polyhydroxybutyrate-Hydroxyvalerate (PHB-HV)/Maize Starch Blend Films. J. Food Eng. 2008, 89 (4), 361–369. https://doi.org/10.1016/j.jfoodeng.2008.04.022.
(40) Biodegradable Polymers for Industrial Applications; Smith, R., Ed.; 2005.
(41) Thellen, C.; Coyne, M.; Froio, D.; Auerbach, M.; Wirsen, C.; Ratto, J. A. A Processing, Characterization and Marine Biodegradation Study of Melt-Extruded
Polyhydroxyalkanoate (PHA) Films. J. Polym. Environ. 2008, 16 (1), 1–11. https://doi.org/10.1007/s10924-008-0079-6.
(42) Wu, C. S. Assessing Biodegradability and Mechanical, Thermal, and Morphologica l Properties of an Acrylic Acid-Modified Poly(3-Hydroxybutyric Acid)/Wood Flours Biocomposite. J. Appl. Polym. Sci. 2006, 102 (4), 3565–3574.
https://doi.org/10.1002/app.24817.
(43) Sanchez-Safont, E. L.; Gonzalez-Ausejo, J.; Gamez-Perez, J.; Lagaron, J. M.; Cabedo, L.
172
Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate)/ Purified Cellulose Fiber Composites by Melt Blending: Characterization and Degradation in Composting Conditions. J. Renew.
Mater. 2016, 4 (2), 123–132. https://doi.org/10.7569/JRM.2015.634127.
(44) Jeguirim, M.; Dorge, S.; Loth, A.; Trouvé, G. Devolatilization Kinetics of Miscanthus Straw from Thermogravimetric Analysis. Int. J. Green Energy 2010, 7 (2), 164–173. https://doi.org/10.1080/15435071003673641.
(45) Lyu, G.; Wu, S. Analytical Pyrolysis Studies of Corn Stalk and Its Three Main Components by TG-MS and Py-GC/MS. J. Anal. Appl. Pyrolysis 2012, 97 (September), 11–18.
https://doi.org/10.1016/j.jaap.2012.04.010.
(46) Baxter, X. C.; Darvell, L. I.; Jones, J. M.; Barraclough, T.; Yates, N. E.; Shield, I. Miscanthus Combustion Properties and Variations with Miscanthus Agronomy. Fuel 2014, 117 (PART A), 851–869. https://doi.org/10.1016/j.fuel.2013.09.003.
(47) 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. https://doi.org/10.1007/s10924-017-1128-9.
(48) Snowdon, M. R.; Mohanty, A. K.; Misra, M. Miscibility and Performance Evaluation of Biocomposites Made from Polypropylene/Poly(Lactic Acid)/Poly(Hydroxybutyrate-
Cohydroxyvalerate) with a Sustainable Biocarbon Filler. ACS Omega 2017, 2 (10), 6446–6454. https://doi.org/10.1021/acsomega.7b00983.
(49) Wu, C. S.; Liao, H. T. The Mechanical Properties, Biocompatibility and Biodegradabil ity of Chestnut Shell Fibre and Polyhydroxyalkanoate Composites. Polym. Degrad. Stab. 2014,
99 (1), 274–282. https://doi.org/10.1016/j.polymdegradstab.2013.10.019.
(50) Greene, J. Biodegradation of Biodegradable and Compostable Plastics under Industria l
Compost, Marine and Anaerobic Digestion. Ecol. Pollut. Environ. Sci. 2018, 1 (1), 13–18.
(51) Manna, A.; Paul, A. K. Degradation of Microbial Polyester Poly(3-Hydroxybutyrate) in Environmental Samples and in Culture. Biodegradation 2000, 11 (5), 323–329. https://doi.org/10.1023/A:1011162624704.
(52) Wu, C. S.; Liao, H. T.; Cai, Y. X. Characterisation, Biodegradability and Application of
Palm Fibre-Reinforced Polyhydroxyalkanoate Composites. Polym. Degrad. Stab. 2017, 140, 55–63. https://doi.org/10.1016/j.polymdegradstab.2017.04.016.
173
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.
174
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.
176
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 Materials and Methods
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 Thermal Properties
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.
𝑋𝐶 = (∆𝐻𝑚
∆𝐻𝑚0 × 𝑤𝑓
) × 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.
5.2.5 Mechanical Properties
5.2.5.1 Tensile and Flexural Properties
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.
5.2.6 Scanning Electron Microscopy (SEM)
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.
5.2.8 Marine Biodegradation
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
5.3 Results and Discussion
5.3.1 Mechanical Properties
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
5.3.7 Marine Biodegradation
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.
5.5 References
(1) Nova Institute. European Bioplastics https://www.european-bioplastics.org/ (accessed Mar
11, 2020).
(2) Gironi, F.; Piemonte, V. Bioplastics and Petroleum-Based Plastics: Strengths and
Weaknesses. Energy Sources, Part A Recover. Util. Environ. Eff. 2011, 33 (21), 1949–1959. https://doi.org/10.1080/15567030903436830.
(3) Jambeck, J.; Geyer, R.; Wilcox, C.; Siegler, T. R.; Perryman, M.; Andrady, A.; Narayan, R.; Law, K. L. Plastic Waste Inputs from Land into the Ocean. 2015, 347 (6223).
https://doi.org/10.1126/science.1260352.
(4) Wilcox, C.; Van Sebille, E.; Hardesty, B. D.; Estes, J. A. Threat of Plastic Pollution to
Seabirds Is Global, Pervasive, and Increasing. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (38), 11899–11904. https://doi.org/10.1073/pnas.1502108112.
(5) Lucas, N.; Bienaime, C.; Belloy, C.; Queneudec, M.; Silvestre, F.; Nava-Saucedo, J. E. Polymer Biodegradation: Mechanisms and Estimation Techniques - A Review.
Chemosphere 2008, 73 (4), 429–442. https://doi.org/10.1016/j.chemosphere.2008.06.064.
189
(6) Kijchavengkul, T.; Auras, R.; Rubino, M.; Selke, S.; Ngouajio, M.; Fernandez, R. T. Biodegradation and Hydrolysis Rate of Aliphatic Aromatic Polyester. Polym. Degrad. Stab.
2010, 95 (12), 2641–2647. https://doi.org/10.1016/j.polymdegradstab.2010.07.018.
(7) Biodegradable Polymers for Industrial Applications; Smith, R., Ed.; 2005.
(8) van den Oever, M.; Molenveld, K.; van der Zee, M.; Bos, H. Bio-Based and Biodegradable
Plastics : Facts and Figures : Focus on Food Packaging in the Netherlands; 2017. https://doi.org/10.18174/408350.
(9) Puls, J.; Wilson, S. A.; Hölter, D. Degradation of Cellulose Acetate-Based Materials: A Review. J. Polym. Environ. 2011, 19 (1), 152–165. https://doi.org/10.1007/s10924-010-
0258-0.
(10) Muniyasamy, S.; Ofosu, O.; John, M. J.; Anandjiwala, R. D. Mineralization of Poly(Lactic
Acid) (PLA), Poly(3-Hydroxybutyrate-Co-Valerate) (PHBV) and PLA/PHBV Blend in Compost and Soil Environments. J. Renew. Mater. 2016, 4 (2), 133–145.
https://doi.org/10.7569/JRM.2016.634104.
(11) Steinbüchel, A.; Valentin, H. E. Diversity of Bacterial Polyhydroxyalkanoic Acids. FEMS
Microbiol. Lett. 1995, 128 (3), 219–228. https://doi.org/10.1016/0378-1097(95)00125-O.
(12) Wang, S.; Lydon, K. A.; White, E. M.; Grubbs, J. B.; Lipp, E. K.; Locklin, J.; Jambeck, J.
R. Biodegradation of Poly(3-Hydroxybutyrate- Co-3-Hydroxyhexanoate) Plastic under Anaerobic Sludge and Aerobic Seawater Conditions: Gas Evolution and Microbia l
Diversity. Environ. Sci. Technol. 2018, 52 (10), 5700–5709. https://doi.org/10.1021/acs.est.7b06688.
(13) Chen, H. Assessment of Biodegradation in Different Environmental Compartments of Blends and Composites Based on Microbial Poly ( Hydroxyalkanoate )S, 2012.
(14) Braunegg, G.; Lefebvre, G.; Genser, K. F. Polyhydroxyalkanoates, Biopolyesters from Renewable Resources: Physiological and Engineering Aspects. J. Biotechnol. 1998, 65 (2–
3), 127–161. https://doi.org/10.1016/S0168-1656(98)00126-6.
(15) Wu, C. S. Assessing Biodegradability and Mechanical, Thermal, and Morphologica l
Properties of an Acrylic Acid-Modified Poly(3-Hydroxybutyric Acid)/Wood Flours Biocomposite. J. Appl. Polym. Sci. 2006, 102 (4), 3565–3574.
https://doi.org/10.1002/app.24817.
(16) Vroman, I.; Tighzert, L. Biodegradable Polymers. Materials (Basel). 2009, 2 (2), 307–344.
https://doi.org/10.3390/ma2020307.
(17) Thellen, C.; Coyne, M.; Froio, D.; Auerbach, M.; Wirsen, C.; Ratto, J. A. A Processing,
Characterization and Marine Biodegradation Study of Melt-Extruded Polyhydroxyalkanoate (PHA) Films. J. Polym. Environ. 2008, 16 (1), 1–11.
https://doi.org/10.1007/s10924-008-0079-6.
190
(18) Arcos-Hernandez, M. V.; Laycock, B.; Pratt, S.; Donose, B. C.; Nikolič, M. A. L.; Luckman, P.; Werker, A.; Lant, P. A. Biodegradation in a Soil Environment of Activated
Sludge Derived Polyhydroxyalkanoate (PHBV). Polym. Degrad. Stab. 2012, 97 (11), 2301–2312. https://doi.org/10.1016/j.polymdegradstab.2012.07.035.
(19) Weng, Y. X.; Wang, X. L.; Wang, Y. Z. Biodegradation Behavior of PHAs with Different Chemical Structures under Controlled Composting Conditions. Polym. Test. 2011, 30 (4),
372–380. https://doi.org/10.1016/j.polymertesting.2011.02.001.
(20) Doi, Y.; Kanesawa, Y.; Tanahashi, N.; Kumagai, Y. Biodegradation of Microbial Polyesters
in the Marine Environment. Polym. Degrad. Stab. 1992, 36 (2), 173–177. https://doi.org/10.1016/0141-3910(92)90154-W.
(21) Kasuya, K.; Takagi, K.; Ishiwatari, S.; Yoshida, Y.; Doi, Y. Biodegradabilities of Various Aliphatic Polyesters in Natural Waters. Polym. Degrad. Stab. 2002, 59 (1–3), 327–332.
https://doi.org/10.1016/s0141-3910(97)00155-9.
(22) Changwichan, K.; Silalertruksa, T.; Gheewala, S. H. Eco-Efficiency Assessment of Bioplastics Production Systems and End-of-Life Options. Sustain. 2018, 10 (4), 1–15. https://doi.org/10.3390/su10040952.
(23) Wu, C. S.; Liao, H. T.; Cai, Y. X. Characterisation, Biodegradability and Application of Palm Fibre-Reinforced Polyhydroxyalkanoate Composites. Polym. Degrad. Stab. 2017,
140, 55–63. https://doi.org/10.1016/j.polymdegradstab.2017.04.016.
(24) Schrader, J. A.; McCabe, K. G.; Grewell, D.; Graves, W. R. Bioplastics and Biocomposite s for Sustainable Horticultural Containers: Performance and Biodegradation in Home Compost. Acta Hortic. 2017, 1170, 1101–1108.
https://doi.org/10.17660/ActaHortic.2017.1170.142.
(25) Seggiani, M.; Cinelli, P.; Balestri, E.; Mallegni, N.; Stefanelli, E.; Rossi, A.; Lardicci, C.;
Lazzeri, A. Novel Sustainable Composites Based on Poly(Hydroxybutyrate-Co-Hydroxyvalerate) and Seagrass Beach-CAST Fibers: Performance and Degradability in
Marine Environments. Materials (Basel). 2018, 11 (5). https://doi.org/10.3390/ma11050772.
(26) Zarrinbakhsh, N.; Mohanty, A. K.; Misra, M. Fundamental Studies on Water-Washing of the Corn Ethanol Coproduct (DDGS) and Its Characterization for Biocomposite
Applications. Biomass and Bioenergy 2013, 55, 251–259. https://doi.org/10.1016/j.biombioe.2013.02.016.
(27) Kasarda, D. D.; Black, D. R. Thermal Degradation of Proteins Studied by Mass Spectrometry. Biopolymers 1968, 6 (7), 1001–1004.
https://doi.org/10.1002/bip.1968.360060712.
(28) van Heeswijk, W. C.; Westerhoff, H. V.; Boogerd, F. C. Nitrogen Assimilation in
Escherichia Coli: Putting Molecular Data into a Systems Perspective. Microbiol. Mol. Biol.
191
Rev. 2013, 77 (4), 628–695. https://doi.org/10.1128/mmbr.00025-13.
(29) Bernhard, A. The Nitrogen Cycle: Processes, Players, and Human Impact. Nat. Educ. 2010, 2 (2), 1–8.
(30) Fuerst, J. A.; Sagulenko, E. Protein Uptake by Bacteria. Commun. Integr. Biol. 2010, 3 (6), 572–575. https://doi.org/10.4161/cib.3.6.13061.
(31) Nagatani, H.; Shimizu, M.; Valentine, R. C. The Mechanism of Ammonia Assimilation in Nitrogen Fixing Bacteria. Arch. Mikrobiol. 1971, 79 (2), 164–175.
https://doi.org/10.1007/BF00424923.
(32) Kratsch, H. A.; Schrader, J. A.; McCabe, K. G.; Srinivasan, G.; Grewell, D.; Graves, W. R. Performance and Biodegradation in Soil of Novel Horticulture Containers Made from Bioplastics and Biocomposites. Horttechnology 2015, 25 (1), 119–131.
https://doi.org/10.21273/horttech.25.1.119.
(33) Javadi, A.; Srithep, Y.; Lee, J.; Pilla, S.; Clemons, C.; Gong, S.; Turng, L. S. Processing
and Characterization of Solid and Microcellular PHBV/PBAT Blend and Its RWF/Nanoclay Composites. Compos. Part A Appl. Sci. Manuf. 2010, 41 (8), 982–990.
https://doi.org/10.1016/j.compositesa.2010.04.002.
(34) Scandola, M.; Focarete, M. L.; Adamus, G.; Sikorska, W.; Baranowska, I.; Świerczek, S.;
Gnatowski, M.; Kowalczuk, M.; Jedliński, Z. Polymer Blends of Natural Poly(3-Hydroxybutyrate- Co -3-Hydroxyvalerate) and a Synthetic Atactic Poly(3-
Hydroxybutyrate). Characterization and Biodegradation Studies. Macromolecules 1997, 30 (9), 2568–2574. https://doi.org/10.1021/ma961431y.
(35) Enriquez, E.; Mohanty, A. K.; Misra, M. Biobased Blends of Poly(Propylene Carbonate) and Poly(Hydroxybutyrate-Co-Hydroxyvalerate): Fabrication and Characterization. J. Appl. Polym. Sci. 2017, 134 (5), 1–10. https://doi.org/10.1002/app.44420.
(36) Zarrinbakhsh, N.; Misra, M.; Mohanty, A. K. Biodegradable Green Composites from
Distillers Dried Grains with Solubles (DDGS) and a Polyhydroxy(Butyrate-Co-Valerate ) (PHBV)-Based Bioplastic. Macromol. Mater. Eng. 2011, 296 (11), 1035–1045. https://doi.org/10.1002/mame.201100039.
(37) U.S. Grains Council. A Guide to Distiller’s Dried Grains with Solubles (DDGS), Third
Edit.; U.S. Grains Council, 2012.
(38) Solle, M. A.; Arroyo, J.; Burgess, M. H.; Warnat, S.; Ryan, C. A. Value-Added Composite
Bioproducts Reinforced with Regionally Significant Agricultural Residues. Compos. Part A Appl. Sci. Manuf. 2019, 124 (April), 105441. https://doi.org/10.1016/j.compositesa.2019.05.009.
(39) Deroiné, M.; Le Duigou, A.; Corre, Y. M.; Le Gac, P. Y.; Davies, P.; César, G.; Bruzaud,
S. Seawater Accelerated Ageing of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate ).
192
Polym. Degrad. Stab. 2014, 105 (1), 237–247. https://doi.org/10.1016/j.polymdegradstab.2014.04.026.
(40) Ndiaye, D.; Gueye, M.; Badji, A.; Thiandoume, C.; Dasylva, A.; Tidjani, A. Effects of
Reinforcing Fillers and Coupling Agents on Performances of Wood–Polymer Composites. In Bio-Based Composites for High-Performance Materials; CRC Press, 2014; pp 113–132. https://doi.org/10.1201/b17601-8.
(41) Lu, H. Processing and Characterization of Bio-Based Composites, Iowa State University, 2014. https://doi.org/10.31274/etd-180810-2811.
(42) Sanchez-Safont, E. L.; Gonzalez-Ausejo, J.; Gamez-Perez, J.; Lagaron, J. M.; Cabedo, L.
Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate)/ Purified Cellulose Fiber Composites by Melt Blending: Characterization and Degradation in Composting Conditions. J. Renew. Mater. 2016, 4 (2), 123–132. https://doi.org/10.7569/JRM.2015.634127.
(43) Jeguirim, M.; Dorge, S.; Loth, A.; Trouvé, G. Devolatilization Kinetics of Miscanthus Straw
from Thermogravimetric Analysis. Int. J. Green Energy 2010, 7 (2), 164–173. https://doi.org/10.1080/15435071003673641.
(44) Lyu, G.; Wu, S. Analytical Pyrolysis Studies of Corn Stalk and Its Three Main Components by TG-MS and Py-GC/MS. J. Anal. Appl. Pyrolysis 2012, 97 (September), 11–18. https://doi.org/10.1016/j.jaap.2012.04.010.
(45) Mørup, A. J.; Christensen, P. R.; Aarup, D. F.; Dithmer, L.; Mamakhel, A.; Glasius, M.;
Iversen, B. B. Hydrothermal Liquefaction of Dried Distillers Grains with Solubles: A Reaction Temperature Study. Energy and Fuels 2012, 26 (9), 5944–5953. https://doi.org/10.1021/ef3008163.
(46) FAO. Food energy - methods of analysis and conversion factors http://www.fao.org/3/y5022e/y5022e03.htm#bm3 (accessed Feb 11, 2020).
(47) Snowdon, M. R.; Mohanty, A. K.; Misra, M. Miscibility and Performance Evaluation of
Biocomposites Made from Polypropylene/Poly(Lactic Acid)/Poly(Hydroxybutyrate -Cohydroxyvalerate) with a Sustainable Biocarbon Filler. ACS Omega 2017, 2 (10), 6446–6454. https://doi.org/10.1021/acsomega.7b00983.
(48) Zarrinbakhsh, N.; Mohanty, A. K.; Misra, M. Improving the Interfacial Adhesion in a New
Renewable Resource-Based Biocomposites from Biofuel Coproduct and Biodegradab le Plastic. J. Mater. Sci. 2013, 48 (17), 6025–6038. https://doi.org/10.1007/s10853-013-7399-1.
(49) Greene, J. Biodegradation of Biodegradable and Compostable Plastics under Industria l Compost, Marine and Anaerobic Digestion. Ecol. Pollut. Environ. Sci. 2018, 1 (1), 13–18.
(50) Deroiné, M.; César, G.; Le Duigou, A.; Davies, P.; Bruzaud, S. Natural Degradation and
Biodegradation of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) in Liquid and Solid
193
Marine Environments. J. Polym. Environ. 2015, 23 (4), 493–505. https://doi.org/10.1007/s10924-015-0736-5.
(51) Manna, A.; Paul, A. K. Degradation of Microbial Polyester Poly(3-Hydroxybutyrate) in
Environmental Samples and in Culture. Biodegradation 2000, 11 (5), 323–329. https://doi.org/10.1023/A:1011162624704.
(52) Ramsay, B. A.; Langlade, V.; Carreau, P. J.; Ramsay, J. A. Biodegradability and Mechanical Properties of Poly-(β-Hydroxybutyrate- Co-β-Hydroxyvalerate)-Starch Blends. Appl. Environ. Microbiol. 1993, 59 (4), 1242–1246.
(53) Madbouly, S. A.; Schrader, J. A.; Srinivasan, G.; Liu, K.; McCabe, K. G.; Grewell, D.;
Graves, W. R.; Kessler, M. R. Biodegradation Behavior of Bacterial-Based Polyhydroxyalkanoate (PHA) and DDGS Composites. Green Chem. 2014, 16 (4), 1911–1920. https://doi.org/10.1039/c3gc41503a.
(54) Gunning, M. A.; Geever, L. M.; Killion, J. A.; Lyons, J. G.; Higginbotham, C. L.
Mechanical and Biodegradation Performance of Short Natural Fibre Polyhydroxybutyra te Composites. Polym. Test. 2013, 32 (8), 1603–1611. https://doi.org/10.1016/j.polymertesting.2013.10.011.
194
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.
195
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.
196
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.
197
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.