Biodegradation of Polycaprolactone Bioplastic in Comparision with Other Bioplastics and Its Impact on Biota

BIODEGRADATION OF POLYCAPROLACTONE BIOPLASTIC IN COMPARISION WITH OTHER BIOPLASTICS AND ITS IMPACT ON BIOTA

A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health

2019

Asma S. Al Hosni

School of Biological Sciences Infection, Immunity and Respiratory Medicine

Table of Contents

List of figures...... 6 List of tables ...... 9 List of abbreviations ...... 10 Abstract ...... 12 Declaration ...... 14 Copyright statement...... 14 Research contributions ...... 16 Acknowledgements ...... 17 Dedication ...... 18 Chapter 1 ...... 19 Introduction ...... 19 1.1 Overview ...... 20 1.2 Benefits and applications of plastics ...... 21 1.3 Problems associated with conventional petrochemical plastics ...... 23 1.4 Biodegradable polymers...... 25 1.4.1 Polycaprolactone...... 27 1.4.2 Polyhydroxybutyrate ...... 28 1.4.3 Polybutylene succinate ...... 31 1.4.4 Polylactic acid ...... 32 1.5 Biological degradation of biodegradable polymers ...... 33 1.6 Advantages and applications of biodegradable polymers ...... 36 1.7 Environmental impact of the degradation process on biota and its quantifications ...... 38 1.8 Thesis aims and objectives ...... 41 1.9 References ...... 43 Chapter 2 ...... 50 A comparative study on the rate of microbial degradation of four biodegradable polymers in soil and compost ...... 50 2.1 Abstract ...... 51 2.2 Introduction ...... 52 2.3 Materials and methods ...... 53 2.3.1 Plastic materials ...... 53 2.3.2 Soil and compost ...... 53 2

2.3.3 Biodegradation of polymer discs under controlled conditions ...... 53 2.3.4 Biodegradation of polymer discs under environmental conditions ...... 54 2.3.5 Isolation and identification of fungal growth on the surface of the polymers under controlled conditions ...... 54 2.3.6 Genomic DNA extraction from fungal mycelia ...... 55 2.3.7 DNA amplification of fungal isolates ...... 55 2.3.8 Purification, sequencing and identification ...... 56 2.3.9 Degradation ability of fungal isolates on PCL strips ...... 56 2.3.10 Tensile strength measurement ...... 56 2.3.11 Scanning Electron Microscopy ...... 57 2.3.12 Statistical analysis ...... 57 2.4 Results ...... 57 2.4.1 Soil and compost analysis ...... 57 2.4.2 Weight change of polymer discs buried under controlled conditions ...... 57 2.4.3 Degradation of polymers buried in soil under environmental conditions .... 63 2.4.4 Changes in physical appearance of the polymers buried in soil under environmental conditions ...... 66 2.4.5 Identification of polymer degrading fungi recovered from the surface of the polymers buried under controlled conditions ...... 67 2.4.6 Biodegradability test ...... 70 2.4.7 Scanning electron microscopy ...... 71 2.5 Discussion ...... 72 2.6 Conclusion ...... 78 2.7 References ...... 80 Chapter 3 ...... 84 Characterisation of polycaprolactone as a promising biodegradable polymer...... 84 3.1 Abstract ...... 85 3.2 Introduction ...... 86 3.3 Materials and Methods ...... 87 3.3.1 PCL strip compost incubation and tensile strength measurement over time 87 3.3.2 PCL powder compost incubation and remaining residual measurement over time 88 3.4 Results ...... 89

3.4.1 PCL strip compost incubation and tensile strength measurements over time 89 3.4.2 PCL powder compost incubation and remaining residual measurements over time. 92 3.5 Discussion ...... 95 3.6 Conclusion ...... 97 3.7 References ...... 98 Chapter 4 ...... 100 The impact of polycaprolactone degradation on microbial communities in compost at different temperatures using next generation sequencing ...... 100 4.1 Abstract ...... 101 4.2 Introduction ...... 102 4.3 Materials and Methods ...... 103 4.3.1 Compost preparation ...... 103 4.3.2 PCL /compost mixture preparation ...... 103 4.3.3 DNA extraction from compost ...... 103 4.3.4 Next generation sequencing and data analysis ...... 104 4.4 Results ...... 106 4.5 Discussion ...... 120 4.6 Conclusion ...... 126 4.7 References ...... 127 Chapter 5 ...... 131 The effect of polycaprolactone degradation in compost on seed germination ...... 131 5.1 Abstract ...... 132 5.2 Introduction ...... 133 5.3 Materials and Methods ...... 134 5.3.1 Compost analysis ...... 134 5.3.2 Compost preparation with 10% PCL ...... 134 5.3.3 Compost preparation with different PCL concentrations ...... 134 5.3.4 Compost extract preparation ...... 134 5.3.5 Cress seed germination ...... 135 5.3.6 Germination of different seed types in compost with 10 % PCL at 55°C. ...135 5.4 Results ...... 135 5.4.1 Cress seed germination in compost extract with 10% PCL concentration ..136

5.4.2 Cress seed germination in compost extract with different PCL concentrations ...... 138 5.4.3 Germination of different seed types in response to 10% PCL compost extract 140 5.5 Discussion ...... 141 5.6 Conclusion ...... 143 5.7 References ...... 144 Chapter 6 ...... 146 General conclusion and future work...... 146 6.1 Summary ...... 147 6.2 Chapter 2. A comparative study on the rate of microbial degradation of four biodegradable polymers in soil and compost...... 147 6.3 Chapter 3: Characterisation of polycaprolactone as a promising biodegradable polymer...... 148 6.4 Chapter 4: The impact of polycaprolactone degradation on microbial communities in compost at different temperatures using next generation sequencing 149 6.5 Chapter 5: The effect of polycaprolactone degradation in compost on seed germination ...... 150 6.6 General discussion and contribution of this study to the field of knowledge ....151 6.7 References ...... 154

Total word count 33,792 (excluding references)

List of figures

Figure Page

1.1 The inter relationship between biodegradable plastics and bio-based plastics. 26

2.1 Percentage weight change over time of PCL discs buried in compost or soil. 59

2.2 Percentage weight change over time of PHB discs buried in compost or soil. 60

2.3 Percentage weight change over time of PLA discs buried in compost or soil. 61

2.4 Percentage weight change over time of PBS discs buried in compost or soil. 62

2.5 Mean of weight over time of PCL polymer buried in soil under environmental conditions. 64

2.6 Mean of weight over time of PHB polymer buried in soil under environmental conditions. 64

2.7 Mean of weight over time of PBS polymer buried in soil under environmental conditions. 65

2.8 Mean of weight over time of PLA polymer buried in soil under environmental conditions. 65

2.9 Changes in physical appearance of polymer discs with time under environmental conditions for (A) PCL, (B) PHB, (C ) PBS and (D) PLA . 66

2.10 Ability of fungal strains isolated from the surface of PCL in compost at 50°C to degrade PCL strips. 70

2.11 Fungal growth on the surface of PCL discs under controlled conditions visualised using SEM. 71

2.12 Fungal growth on the surface of PLA discs under controlled conditions visualised using SEM. 71

2.13 Fungal growth on the surface of PBS discs under controlled conditions visualised using SEM. 72

2.14 Fungal growth on the surface of PHB discs under controlled conditions visualised using SEM. 72

3.1 Tensile strength measurements of PCL strips over time at 25°C 89

3.2 Tensile strength measurements of PCL strips over time at 37°C. 90

3.3 Tensile strength measurements of PCL strips over time at 45°C. 91

3.4 Tensile strength measurements of PCL strips over time at 50°C. 91

3.5 Mean percentage of PCL residual remaining with time at 25°C. 93

3.6 Mean percentage of PCL residual remaining with time at 37°C. 93

3.7 Mean percentage of PCL residual remaining with time at 45°C. 94

3.8 Mean percentage of PCL residual remaining with time at 50°C. 94

3.9 Mean percentage of PCL residual remaining with time at 55°C. 95

4.1 Linear regression analysis of the correlation between temperature and number of OTUs. 107

4.2 Rarefaction curves of observed fungal OTUs richness for compost at different temperatures at 97% sequence similarity. 109

4.3 Principal Component Analysis (PCA) of fungal communities isolated from initial compost (Time 0 control) and compared with compost containing 10% PCL and compost control at Week 5 and 8 at five different temperatures. 110

4.4 Relative abundance of fungal sequences (order level) from compost samples with (10%) PCL and compost control at two time points (Weeks 5 and 8) at 25°C compared with the initial compost (time 0 control) at ambient temperature. 112

4.5 Relative abundance of fungal sequences (order level) from compost samples with (10%) PCL and compost control at two time points (Weeks 5 and 8) at 37°C compared with the initial compost. 113

4.6 Relative abundance of fungal sequences (order level) from compost samples with (10%) PCL and compost control at two time points (Weeks 5 and 8) at 45°C compared with the initial compost. 114

4.7 Relative abundance of fungal sequences (order level) from compost samples with (10%) PCL and compost control at two time points (Weeks 5 and 8) at 50°C and compared with the initial compost. 115

4.8 Relative abundance of fungal sequences (order level) from compost samples with (10%) PCL and compost control at two time points (Weeks 5 and 8) at 55°C and compared with the initial compost. 116

4.9 Relative abundance of fungal sequences for the highest dominant taxa orders present in compost samples at all five temperatures. 117

4.10 Number of reads of fungal sequences of genus levels below Eurotiales order for all compost samples that used in this study. 118

4.11 Number of reads of fungal sequences of genus levels below 119

Hypocreales order for all compost samples used in this study.

4.12 Number of reads of fungal sequences of genus levels below Sordariales order for all compost samples used in this study. 120

5.1 Cress seeds tested in compost extract from compost with 10% PCL at five different temperatures in comparison to control compost. 137

5.2 Cress seeds tested in compost extract with different PCL concentrations under three relatively high temperatures in comparison to control compost. 139

5.3 Germination of wild rocket, mustard and lettuce seeds in compost extract prepared at 55°C in 10% PCL in comparison to control compost. 140

List of tables

Table Page

1.1 Applications of some widely used synthetic plastics. 22

1.2 Applications of some widely used biodegradable polymers. 38

2.1 Summary of percentage weight change of polymers in soil and compost at various temperatures. 63

2.2 Number of morphotypes growing on PDA plates isolated from the surface of PCL at different temperatures. 67

2.3 Number of morphotypes growing on PDA plates isolated from the surface of PHB at different temperatures. 67

2.4 Identification of the putative polymer degraders isolated from the surface of the polymers buried in soil and compost under controlled conditions. 69

4.1 Number of fungal OTUs and the total number of reads calculated for samples incubated at five different temperatures for five or eight weeks. 106

4.2 Diversity indices calculated for all fungal samples incubated at five different temperatures for five or eight weeks. 108

List of abbreviations

BLAST Basic Local Alignment Search Tool

BDPs Biodegradable Polymers

BSA Bovine Serum Albumin

DCM Dichloromethane solvent

DEPC Diethylpyrocarbonate treated water

EDTA Ethylenediaminetetraacetic

GHG Greenhouse Gas

ITS Internal Transcribed Spacer

MAP Modified Atmospheric Storage

NCBI National Centre for Biotechnology Information

NGS Next Generation Sequencing

OTUs Operational Taxonomic Unit

PBS Polybutylene succinate

PCL Polycaprolactone

PE Polyethylene

PHA Polyhydroxyalkanoate

PHB Polyhydroxybutyrate

PLA Polylactic acid

PCR Polymerase Chain Reaction

PP Polypropylene

PS Polystyrene

PVC Polyvinyl chloride

PDA Potato Dextrose Agar

PCA Principal Component Analysis

SEM Scanning Electron Microscopy

SDS Sodium Dodecyl Sulfate

UV Ultra Violet light

WHC Water Holding Capacity

YES Yeast Extract Salts

Abstract

The world is facing many global challenges and one of these is the waste disposal management problem, which arose due to the high production of plastics worldwide. Synthetic plastic waste can accumulate in the environment for decades or even centuries due to their recalcitrant nature. As a way of solving this problem, the production and use of biodegradable polymers (BDPs) have been gradually increased. Naturally, biodegradable polymers decompose into carbon dioxide, water, inorganic compounds, methane and biomass via microbial activity. Polyhydroxybutyrate (PHB), polybutylene succinate (PBS), polylactic acid (PLA), and polycaprolactone (PCL) are examples of the available BDPs. PCL is a synthetic, aliphatic polymer that is compatible with other polymers and has many applications. There have been extensive researches on the microbial degradability of many polymers under different conditions. However, in addition to the biodegradability of these BDPs, the biodegradation rate and the suitable environment for the degradation of each polymer need to be evaluated in order to decide the suitable waste management method. Moreover, the impact of the products from the degradation of BDPs on the environment and their effect on biota require more investigations. Therefore the aims of this research was to (a) determine the rate of microbial degradation of four biodegradable polymers in soil and compost under different conditions, (b) to investigate the degradation of PCL in different forms to determine the characterisation of PCL as a promising biodegradable polymer, (c) to determine the impact of PCL degradation on microbial communities in compost at different temperatures using next generation sequencing, and (d) to determine if PCL degradation has any effect on seed germination in compost. The conditions of the burial environment had a clear and direct effect on the degradation of different BDPs. The degradation of PCL was the fastest and temperature was highly correlated to the rate of PCL degradation under controlled conditions. The degrading microorganisms that were isolated from the surface of the polymers were identified and found that Thermomyces lanuginosus was the main PCL degrader at 50°C in compost. When testing the effect of PCL degradation on microbial community structure it was been found that fugal communities were distributed according to the presence of PCL at each temperature. However, temperature as well was a factor for fungal community structure variation overall. Under most

12 conditions PCL has no adverse effect on the germination of seeds. However, PCL with concentration of 5% and above inhibited cress seed germination when incubated at 55°C.

Declaration

No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of any other university or other institute of learning.

Copyright statement i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trademarks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=24420), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.library.manchester.ac.uk/about/regulations/) and in The University’s policy on Presentation of Theses.

14 iii. The ownership of certain Copyright, patents, designs, trademarks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=487), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’s policy on Presentation of Theses.

Research contributions

Oral Presentation:

 The 3rd International Conference and Exhibition on Biopolymers and Bioplastics, San Antonio, USA (2016). Asma Al Hosni and Geoff Robson. Biodegradation of Biopolymers under soil and compost conditions.  The faculty of Life science PhD conference, Manchester, UK (2016). Asma Al Hosni and Geoff Robson. Microbial degradation of biodegradable polymers

Poster Presentation:

 The International Conference on Sustainable Bioplastics, Alicante, Spain (2016). Asma Al Hosni and Geoff Robson. The effect of temperature on the degradation rate of four different types of biodegradable polymers buried in soil and compost.

 The 17th International Biodeterioration & Biodegradation Symposium (IBBS), Manchester, UK (2017). Asma Al Hosni and Geoff Robson. The effect of temperature on the rate of degradation of four different types of biodegradable polymers buried in soil and compost under controlled laboratory conditions.

 World Congress on Novel Trends and Advances in Biotechnology, Barcelona, Spain (2018). Asma Al Hosni. Poly-caprolactone: a promising biodegradable polymer.

Acknowledgements

In the name of Allah, the most Merciful and Gracious, above all, from the depth of my heart, I thank God Almighty, for having made everything possible by giving me strength, courage and blessing to do this work and completing this thesis.

First of all, I would like to express my gratitude and deep sorrow to my late supervisor Dr Geoffrey Robson who passed away during the write up of this thesis. His support, guidance and kind help has lighted my path to be a good researcher.

Then, I would like to take this opportunity to thank my supervisor Dr Jon Pittman for his support, knowledge, excellent guidance and kind help he has given me at every stage during the write up of this thesis.

I would like to show my appreciation for my colleagues in Robson’s group for their help during my laboratory work and for having a great time together and sharing many experiences. Special thanks go to Zhabiz Vilkiji for her help in NGS data analysis.

Very special thanks for my friends in ‘Manchester Group’. Thank you for your understanding and encouragement in many moments of crises. Thank you for sharing many special moments during this chapter of my life. Thank you for being good friends.

My sincere gratitude goes to my whole family and friends for their continued care, support and encouragement.

I would also like to say a heartfelt ‘thank you’ to my Mum, Dad. There are no words to express my gratitude and thanks to them for their unconditional love, their love has been the major spiritual support in my life.

Finally, I would like to express the deepest gratefulness to my husband, Nasser Al-Housni for his endless support, understanding and love. I would never have been able to be at this level without him. His support, constant encouragement and guidance made me to believe in myself. I also would like to send my love to my three children, Alanood, Mohammed and Omar who shared patiently this entire amazing journey with me.

Dedication

" In the memory of Dr Geoffrey Robson "

Chapter 1

Introduction

1.1 Overview

Over the last six decades, the use of plastic materials has had a major impact on our daily lives. Plastics have become essential for modern societies due to their extensive and diverse range of applications, and their production globally has increased every year (Tokiwa et al., 2009; Emadian et al., 2017). Plastics are defined as a range of different polymers with a high molecular weight that are exploited by mankind in a range of different applications (Tokiwa et al., 2009). The term ‘plastic’ is derived from the Greek word ‘plastikos’, which means an ability to be moulded into different shapes (Shah et al., 2008).

Plastics are synthesized by the polymerization of monomers derived principally from petrochemical sources such as oil and gas with the further addition of different chemical additives. About 4% of gas and oil produced worldwide is used as the raw material for plastics and a similar amount is used as energy in their manufacture (Andrady and Neal, 2009). For these synthetic polymers to be produced there are two main processes: the first process involves breaking down the double bonds in the original olefin to form new carbon – carbon bonds to yield the carbon chain polymers and this is done by additional polymerization. The second process is to eliminate water between a carboxylic acid and an alcohol or amine to form a polyester or polyamide (Zheng et al., 2005).

Plastics first began to substitute natural materials such as wood, leather and metals more than half a century ago (Shah et al., 2008; Sivan, 2011). Since then, the use of plastics has grown year on year and Emadian et al. (2017) have estimated that plastic production globally reached more than 300 million tonnes in 2015. This production is a 3.5% increase as compared to 2014 (Mangaraj et al., 2018).

Currently, plastics are an indispensable and basic material in all aspects of the modern world. The durability and diverse properties of plastics make them ideal materials in the manufacture of a wide range of products (Thompson et al., 2009a). They are inexpensive, strong, lightweight, and flexible (Hopewell et al., 2009; Thompson, et al., 2009b; Sivan, 2011; Muthuraj et al., 2018). Plastics also have high thermal and electrical insulation properties (Thompson et al., 2009a, 2009b). Moreover, many plastics are intrinsically resistant to microbial attack (Shah et al., 2008).

1.2 Benefits and applications of plastics

There are many applications and uses of plastics because of their favourable mechanical and thermal properties. In addition plastics are also cheap to manufacture, stable and durable (Shah et al., 2008).

Plastic is involved in almost all daily aspects of our lives. These include footwear, which depends heavily on plastic, as well as clothing. It has been stated that more than 40 million tonnes of plastics were converted into textile fibre around the world to be used in apparel manufacture (Andrady and Neal, 2009). Plastic also can provide products that can be used for public health applications such as drips, aseptic medical packaging and blister packs for pills (Andrady and Neal, 2009; Thompsonet al., 2009a). Plastic can enable clean drinking water supplies since drinking water can be stored in plastic bottles, while plastics are heavily involved in water control and distribution system (Andrady and Neal, 2009).

In addition, plastics are involved in food production, storage and distribution. For example, plastics can provide a good quality of packed food by prolonging the life of some types of food like meat and vegetables and monitoring the time-temperature history. This is done by building a low-cost indicator label into the packaging (Andrady and Neal, 2009). Furthermore, plastics have characteristics such as good flexibility and tear strength, which is appropriate for food packaging. Moreover, plastics are a good barrier to O2, CO2, anhydride and aroma compounds and for sealing the heat (Tharanathan, 2003; Siracusa et al., 2008).

Due to their light weight, plastics are considered as an ideal material for modern transport systems. This decreases the transportation cost and hence the CO2 emissions in the atmosphere (British Plastic Federation). Transportation vehicles contain around 20% plastics and some aircraft such as the Boeing Dreamliner is designed from approximately 50% plastic as stated by Andrady and Neal, (2009). In addition, the world’s largest commercial aircraft (Airbus A380) contain about 22% of carbon reinforced plastics. This benefits in the reduction of the fuel burn to a rate equivalent to that of economical family car (British Plastic Federation, 2018).

Moreover, plastics play a major role in building constructions. Many building materials are produced from plastic such as insulation materials, windows, water pipes and door frames, thus reducing building costs (Andrady and Neal, 2009).

Another major application of plastics is for packaging due to their light weight and barrier properties. Over a third of plastic consumption is for packaging (Koelmans et al., 2014; Muthuraj et al., 2018). The plastics that are most widely used for packaging are polyethylene (PE), polypropylene (PP), polystyrene (PS), and polyvinyl chloride (PVC) (Shah et al., 2008). Table 1.1 summarises some of the applications and uses for the most commonly used conventional plastics.

Table 1.1 Applications of some widely used synthetic plastics.

Plastic Use Reference

Plastic bags, milk and water (Shah et al., 2008; Mukherjee and Polyethylene bottles, food packaging film, toys, Chatterjee, 2014; Sharma et al., and motor oil bottles 2016)

Disposable cups, packaging (Shah et al., 2008; Mukherjee and Polystyrene materials, laboratory wares and Chatterjee, 2014; Sharma et al., certain electronic uses 2016)

Tires, refrigerator insulation, (Shah et al., 2008; Mukherjee and Polyurethane furniture cushions, sponges and Chatterjee, 2014; Sharma et al., life jackets. 2016)

Bottle caps, drinking straws, (Shah et al., 2008; Mukherjee and medicine bottles, car seats and Polypropylene Chatterjee, 2014; Sharma et al., batteries, disposable syringes and 2016) carpet backings

(Shah et al., 2008; Mukherjee and Polyethylene Soft drink bottles, textile fibres, Chatterjee, 2014; Sharma et al., terephthalate sleeping bag and pillow filling 2016)

Small bearings, speedometer (Shah et al., 2008; Mukherjee and gears, windshield wipers, water Nylon Chatterjee, 2014; Sharma et al., hose nozzles, football helmets, 2016) race horse shoes and cell phones

1.3 Problems associated with conventional petrochemical plastics

Despite the wide range of plastic applications as well as its industrial and societal benefits, the disposal of plastics and wide-scale environmental contamination has led to plastics becoming a major environmental pollutant worldwide (Shah et al., 2008; Pathak et al., 2014). The recalcitrant nature of many plastics means that they can stay in the environment for decades or even centuries. As a consequence, significant amounts of plastics accumulate in the terrestrial and aquatic environment, such as in landfills and in the oceans, resulting in significant environmental pollution and waste management issues (Hopewell et al., 2009; Kumar and Maiti, 2016).

There are huge global concerns about the environmental impact of plastic waste because the disposal systems are scarce. Moreover, toxic waste pollution may occur from the incineration process of plastic wastes and suitable landfills are limited overall. In addition, the petroleum resources from which these plastics are produced are finite and recycling of plastic wastes is expensive (Pathak et al., 2014). It has been estimated that 275 million tonnes of plastic waste was produced in 192 coastal countries in 2010 (Jambeck et al., 2015). In addition, global plastic production reached 250 million tonnes per year (Haider et al., 2019). Although some plastics can be recycled, the majority accumulate in landfills (Thompson et al., 2009b). The most consumed plastic is PE; approximately 140 million tons of these polymers are produced annually. Therefore, any reduction in the accumulation of PE waste alone will contribute to a principal change in the overall reduction of the plastic waste globally (Sivan, 2011).

Most of the plastics being produced are plastic packaging materials, accounting for about 26% of the overall plastic production volume, which are discarded within a year following manufacture (Hopewell et al., 2009; Song et al., 2009). Moreover, it has been predicted that the production of plastic packaging materials will be increased in 2030 and 2050 by two-fold and three-fold, respectively compared to today's global plastic production (Muthuraj et al., 2018). In the UK, it is estimated that 1 million tonnes of non-bottle domestic mixed plastic packaging waste is produced every year and is increasing annually by between 2% and 5% (Song et al., 2009). Additionally, the disposal of plastic material in the environment leads to wide-scale pollution and urban litter. Urban litter comprises different discarded materials, including thermoplastic material, which are not

23 biodegradable and can persist in the environment for long period of time (Andrady and Neal, 2009).

Furthermore, plastics disposed in marine habitats led to the accumulation of very large quantities of plastic debris on remote shorelines and inaccessible areas of the deep sea. It has been shown by Thompson et al. (2009a) that 50-80% of all shoreline debris can be attributed to plastics. In addition to that, there are high levels of microplastic contaminations compared with the global average in the North Pacific Ocean and the adjacent marginal seas (Shim and Thomposon, 2015).

In addition, production and processing of plastics can use huge amount of energy that lead to increased greenhouse gas (GHG) emissions which will contribute to global warming. Furthermore, when burning plastics several toxic gases, which have adverse effects on the environment and on public health will be released, such as carbon monoxide, chlorine, hydrochloric acid, amines, nitrides, styrene, benzene, 1, 3-butadiene, and acetaldehyde (Mangaraj et al., 2018). In addition, combustion of PVC plastics leads to the production of furans and dioxins which are carcinogenic and persistent organic pollutants (Shah et al., 2008).

Another problem associated with conventional petrochemical plastics is the addition of additives. Plastic polymers are often not used alone for the production of plastic materials but are mixed with different additives to enrich their performance. These additives are used extensively and include inorganic fillers, plasticizers, thermal and ultraviolet stabilizers, flame retardants and colourings (Thompson et al., 2009a). However, some of these additives are toxic, for example lead, tributyl citrate and poly(ethylene glycol) are used as additives in PVC (Haider et al., 2019). Other additives like phthalates, bisphenol A and polybrominated diphenyl are plasticisers which have detrimental effects in humans and animals (Thompson et al., 2009a). These can be transferred to humans by different means, for example, directly through plastic toys taken in the mouth by children, or indirectly through the packing materials of food and drinks (Thompson et al., 2009b). It has been shown that a wide range of animals can ingest fragmented plastics in the environment and as a consequence this will lead to the transfer of toxic chemicals to wildlife (Thompson et al., 2009b).

Therefore and due to these growing environmental problems associated with the use and disposal of high amounts of plastics, the adoption of user-friendly and eco-friendly substitutes to conventional plastics are required. As a consequence, biodegradability and the use of biodegradable polymers are not only a functional requirement but also an important environmental attribute (Siracusa et al., 2008). Hence biodegradable polymers are considered as one of the main solutions to the problem of plastic accumulation (Zheng et al., 2005; Tokiwa et al., 2009; Haider et al., 2019).

1.4 Biodegradable polymers

Biodegradable polymers (BDPs) are polymeric materials that can be decomposed into carbon dioxide, methane, water, inorganic compounds or biomass by the enzymes of microorganisms (Song et al., 2009; Laycock et al., 2017; Haider et al., 2019). Another definition of the biodegradable polymers is “polymers, susceptible to degradation by biological activity, with the degradation accompanied by a lowering of its mass” and this definition is according to the International Union of Pure and Applied Chemistry (IUPAC) (Vert et al., 2012). The chains of BDPs can be broken down as well by non-enzymatic processes such as chemical hydrolysis (Laycock et al., 2017). The use of biodegradable polymers can aid in resolving a number of waste management issues as they are degraded ultimately to CO2 and H2O. Therefore, these polymers can be directed to conventional industrial composting systems (Song et al., 2009). A study has indicated that BDPs can be included in aerobic composting or anaerobic digestion combined with methane capture for the generation of energy (Hopewell et al., 2009). Moreover, these BDPs can act as a substitution to conventional materials because they are more sustainable, renewable and more eco-friendly in nature. In addition these materials are also biodegradable, biocompatible (not harmful or toxic to living tissue) and hence can be used in many different applications (Swain et al., 2018).

The term bioplastics refers to green plastics and this terminology could be confusing. Bioplastics can be divided into two categories: (Mohanty et al., 2000; Kuruppalil, 2011; Pathak et al., 2014; Emadian et al., 2017).

 Bio-based plastics; these are plastics that are synthesized from biomass and renewable resources such as cellulose, corn, sugarcane and bacteria. These plastics

are considered as natural polymers. Examples of these are polylactic acid (PLA) and polyhydroxyalkanoate (PHA).  Biodegradable plastics; these are plastics synthesized from non-renewable resources such as petroleum and gas. These plastics are considered as synthetic polymers and examples of these are polycaprolactone (PCL) and polybutylene succinate (PBS).

It is important to understand that not all bio-based plastics are biodegradable and vice versa. For example, bio-based PE is produced from renewable resources but is not biodegradable. (Emadian et al., 2017; Rujnić-Sokele and Pilipović, 2017; Dilkes-Hoffman et al., 2019). Figure 1.1 shows the inter relationship between biodegradable plastics and bio- based plastics (Tokiwa et al., 2009).

Figure 1.1 The inter relationship between biodegradable plastics and bio-based plastics (Tokiwa et al., 2009).

PCL and PBS are petroleum based polymers but can undergo microbial degradation. Polyhydroxubutyrate (PHB), PLA and starch blends are made up from biomass from renewable resources and are biodegradable (Tokiwa et al., 2009; Muthuraj et al., 2018). However, it is worth highlighting that biodegradability is not only a function of origin but also of chemical structure and degrading environment (Mohanty et al., 2000; Pathak et al., 2014).

The global production of bioplastics has reached 2.11 million tonnes in 2018 and is expected to reach 2.62 million tonnes in 2023 according to the European Bioplastics Association (European Bioplastics, 2018). Some biodegradable plastics such as PHA, PCL, polylactides, aliphatic polyesters, polysaccharides and copolymer or blends of these

26 materials have become established over the last few years because of their similar properties to conventional plastics (Shah et al., 2008). A study stated that the production of bioplastics increased from 1.6 to 2.0 million tonnes during the period 2013–2015 (Mangaraj et al., 2018). Biodegradable polymers are now be produced on an industrial scale; however, these polymers are more expensive than the conventional synthetic polymers. Their production is low compared to conventional plastics and currently accounts for less than one per cent of plastic production world-wide (Thompson et al., 2009a).

Overall there is a need for replacement of the conventional plastics with bioplastics because the production of conventional plastics can consume 65% more energy. This production is unsustainable because it can cause environmental pollution and emits 30 – 80% more GHG than bioplastics (Mangaraj et al., 2018).

1.4.1 Polycaprolactone

PCL is a biodegradable synthetic polymer that can be degraded by microorganisms and is derived from the chemical synthesis of crude petroleum (Shimao, 2001; Woodruff and Hutmacher, 2010). It is a partially-crystalline aliphatic polyester with a low melting point (60°C) and glass transition temperature of -60 °C. In addition, PCL is a thermoplastic polymer, which has chemical resistance to water, oil and chlorine. The low melting point of PCL makes it suitable for composting, which will help in the disposal of this polymer. Moreover, PCL has low viscosity and it is easy to process because it can be soluble in a wide range of organic solvents (Nair and Laurencin, 2007; Funabashi et al., 2009). The molecular formula of PCL is (C6H10O2)n and it is synthesized by ring-opening polymerization of the cyclic monomer Ɛ-caprolactone (Tokiwa et al., 2009; Luyt and Malik, 2019). There are many applications of PCL especially in medicine and agricultural fields. For example PCL can be used as release systems for drugs (Imre and Pukanszky,2013). Moreover, this polymer can be used as agricultural mulch and other films, and seedling containers (shah et al., 2008; Imre and Pukanszky,2013). If this polymer is mixed with starch, it can then be used as rubbish bags (Siracusa et al., 2008). This polymer is composed of a 6-hydroxyhexanoate backbone, which can be attacked by microorganisms and undergo biodegradation (Sabev et al., 2006). The chemical structure of PCL is:

([-OCH2CH2CH2CH2CH2CO-]n) (Tokiwa et al., 2009).

PCL degrading microorganisms are ubiquitous in the environment. Aerobic and anaerobic microorganisms can act upon the degradation of PCL (Tokiwa et al., 2009). It has been found that proportions of PCL-degrading aerobic and anaerobic microorganism population were estimated to be 0.8 - 11% and 0.6 - 12.9%, respectively (Nishida and Tokiwa, 1993). Many studies have investigated different PCL-degrading microorganisms, for example, Penicillium oxalicum strain DSYD05-1 which was isolated from soil and found to completely degrade high molecular weight PCL after 10 days (Li et al., 2012). Furthermore, an Aspergillus sp. Strain ST-01 that was also isolated from soil was found to degrade PCL completely after 6 days incubation at 50°C under aerobic condition. (Sanchez et al., 2000). Moreover, new species belonging to the genius Clostridium were found to act upon PCL under anaerobic condition (Tokiwa et al., 2009). This polymer can also be degraded by lipases and esterases although little is known about PCL depolymerases (Shimao, 2001). A study by Nishida and Tokiwa (1993) found that cutinases of some fungal phytopathogens can degrade PCL and hence can act as PCL depolymerases. Moreover, it has been shown that a cutinase from a Fusarium sp. is in fact a PCL depolymerase (Shimao, 2001). In another study, it has been investigated that lipases from Rhizopus delemar and Rhizopus arrhizus can promote the degradation of this polymer (Sabev et al., 2006; Pathak and Navneet, 2017). A study investigated that disposing PCL in a bioactive environment such as soil will lead to the degradation of this polymer (Gross and Kalra, 2002). In vitro degradation of PCL has been reported by identified taxa from deep sea environments including by Tenacibaculum, Alcanivorax and Pseudomonas (Sekiguchi et al., 2011).

PCL degradation can take place by at least two stages. In the first stage, non-enzymatic hydrolytic ester cleavage will take place and this will lead to the reduction of molecular weight. In the second stage, the chain scission rate will slow down and lead to weight loss of the polymer and hence the polymer will be susceptible to fragmentation (Leja and Lewandowicz, 2010; Krasowska et al., 2016).

1.4.2 Polyhydroxybutyrate

PHB is a polyester that occurs naturally and accumulates in bacterial cells as a reservoir of carbon and energy (Shimao, 2001). This polymer is synthesised under unbalanced growth conditions (Bonartsev et al., 2007). PHB is a fully biodegradable polymer with good biocompatibility and can be broken down rapidly by microorganisms present in the soil 28 producing 3-hydroxybutyrate, which is a normal mammalian metabolite (Dawes, 1988). The commercial name for this polymer is Biopol (Mohanty et al., 2000). PHB belongs to PHA family which is known to be a bacterial polymer that is produced by several gram-positive and gram-negative bacteria from at least 75 different genera (Reddy et al., 2003). It can also be produced from other types of substrates including renewable resources like starch, cellulose and sucrose or from fossil resources like methane, mineral oil and hard coal. PHB can be produced as well from different by- products such as molasses, whey and glycerol, or from chemicals like propionic acid and from carbon dioxide (Reddy et al., 2003).

The history of this polymer dates back to 1925 when it is first isolated and characterised by Lemoigne at the Pasteur Institute in Paris. Later, extensive studies came up with the conclusion that PHB is stored in the bacterial cells as an energy reservoir in the same way that fat accumulates in mammals (Holmes, 2002). The chemical structure of PHB is

(– { O – CH (CH3) – CH2 – ( C=O ) }n –) (Holmes, 2002).

PHB has many properties that make it as a good alternative to conventional plastics. For example, PHB is considered as a thermoplastic polymer and resembles PP in its physical properties such as melting point (180°C), high degree of crystallinity, and glass-rubber transition temperature (0 - 5°C). However, it is stiffer and more brittle than PP (Holmes, 2002; Chaijamrus and Udpuay, 2008). The degree of brittleness depends on other properties like degree of crystallinity, glass temperature and microstructure. The longer this polymer is stored at room temperature, the more brittle it becomes (Ghaffar, 2002). It is optically active and isotactic with high degree of polymerization (Reddy et al., 2003). In regard to its chemical properties, PHB has resistance to inferior solvents and ultraviolet weathering. It also has good gas barrier properties (Holmes, 2002).

There are a number of potential applications of PHB in different disciplines such as example in medicine where PHB can be used as sutures, surgical mesh and many other applications (Chen and Wu, 2005). Other applications of PHB are in pharmacology where it can be used as microcapsules in drug treatment and for controlled drug release (Dawes, 1988; Holmes, 2002). PHB as well can be used in the food industry and this includes bottles, laminated foil and one-use cups (Ghaffar, 2002).

PHB can be fully degraded and converted to carbon dioxide and energy by the action of microorganisms such as bacteria and fungi. It has been shown that fungi present on the surface of the polymer can excrete extracellular enzymes that solubilise the surface of PHB and then absorb the soluble degradation products for metabolism (Holmes, 2002). The main enzyme responsible for the degradation of PHB is PHB depolymerase. This enzyme has been identified in many organisms including Rhodospirillum rubrum, Bacillus megaterium, Acinetobacter beijerinckii, Pseudomonas lemoignei and Alcaligenes faecalis (Reddy et al., 2003). The degradation of PHB can take place under aerobic (producing carbon dioxide and water) and anaerobic (producing carbon dioxide and methane) conditions (Reddy et al., 2003). Several PHB-degrading microorganisms have been isolated from different natural ecosystems like soil, activated and aerobic sludge and water bodies. Examples of these are Bacillus sp., Pseudomonas sp. and Streptomyces sp. (Tokiwa et al., 2009). It has been shown that in nature, the percentage of PHB- degrading microorganisms was estimated to be 0.5-9.6% of the total microbial biomass and most of these were isolated from ambient or mesophilic temperature, and very few at higher temperatures. A study showed that 90% of PHB film was degraded by a thermotolerant Aspergillus sp. after five days of cultivation at 50°C. Moreover, it has been stated by Siracusa et al. (2008) that PHB is degraded in 5-6 weeks in microbiologically active environments, while under anaerobic conditions, the degradation is faster.

In order to start PHB degradation, PHB depolymerase should be in contact with the polymer then hydrolysis can occur. This will produce oligomers, dimers and monomers, which can then be metabolised by microorganisms to produce biomass depending on the environment where the degradation takes place (Gutierrez-Wing et al., 2011). A study performed a comparison between the hydrolytic and enzymatic degradation processes of PHB films (Kumagai et al., 1992). The results showed that the speed of enzymatic degradation by the PHB depolymerase was faster by two or three orders of magnitude than the speed of simple hydrolytic degradation. This degradation by the PHB depolymerase was found to occur on the surface of the films. It was also reported by Tokiwa et al. (2009) that PHB, as well as PCL and PBS, can be degraded by 39 different bacterial strains of the classes Firmicutes and Proteobacteria. In addition, the literature showed that PHB can be degraded thermally, when the temperature is close to melting point. This thermal degradation occurs exclusively through a random chain scission mechanism involving a six-membered ring transition state (Mohanty et al., 2000). 30

1.4.3 Polybutylene succinate

PBS is linear aliphatic thermoplastic polyester and one of the biodegradable polymers with many favourable properties such as melt processability, and both thermal and chemical resistance. Softness, gas barrier properties, and melt viscosity are other desirable properties of PBS (Kim et al., 2006; Ali and Mohan, 2009; Kumar and Maiti, 2016). PBS has a melting point between 90-120°C and glass transition temperature of - 45°C to -10°C, which is considerably low(Adamopoulou, 2012). The trade name of this polymer is Bionolle (Mohanty et al., 2000; Shibata et al., 2006). This polymer is produced by the condensation reaction of glycols such as 1,4-butanediol and aliphatic dicarboxylic acid such as succinic acid, which are used as principal raw materials (Kim et al., 2005). PBS can be used for several applications due to its excellent properties. There are many applications of this polymer such as packaging films, bags, mulch film, flushable hygiene, food container, textiles and disposable medical devices (Xu and Guo, 2010). The chemical structure of PBS is

([-O(CH2)4OOC(CH2)2CO-]n) (Tokiwa et al., 2009).

PBS is a degradable polymer that can be degraded by naturally occurring microorganisms and enzymes that are extracted from bacteria and fungi, and this degradation will yield

CO2 and H2O (Kim et al., 2005). PBS can also be degraded by hydrolytic degradation. In this degradation the molecular weight of the polymer will decline steadily with time and this is an indication that the hydrolytic degradation is proceeding via random chain scission (Xu and Guo, 2010). Like any other biodegradable polymer, the biodegradation of PBS depends on many factors such as chemical structure, crystallinity, macroscopic shape of the particles and degradation conditions (Adamopoulou, 2012). The molecular weight of the polymer will decrease due to the hydrolysis mechanism that occurs at the ester linkages and thus can be further degraded by microorganisms (shah et al., 2008). A leading Korean manufacturer of PBS has presented data stating that 50% degradation occurs over one month for a 40 µm thick film in garden soil (Shah et al., 2008). It has been shown that pH has a great effect on the rate of hydrolytic degradation of PBS such that the degradation rate would increase with increasing pH (Adamopoulou, 2012). A study showed that PBS can be enzymatically degraded by lipases from Rhizopus sp., Pseudomonas sp., Mucor miehei, Aspergillus niger, and Chromobactrium viscosum (Pranamuda et al., 1995). Moreover, it has been demonstrated that in a soil environment 31 the percentage of PBS-degrading bacteria is about 0.2-6 % of the total biomass and that a bacterial type belonging to the genus Roseateles showed the highest degrading activity of all types that have been examined (Suyama and Tokiwa, 1998). In addition, it has been presented that the degradation of PBS under activated sludge is more rapid than in buried soil (Adamopoulou, 2012). In addition, under different natural water types, the degradation of PBS has been examined and it was found that the biodegradation rate decreases in the following order: seawater from the bay > fresh water from the river > freshwater from the lake > seawater from the Pacific Ocean (Adamopoulou, 2012). Another investigation showed that Amycolatopsis sp. HT-6 can degrade PBS as well as PHB and PCL (Tokiwa et al., 2009). Moreover, it has been presented that after the cultivation of PBS for 8 days in liquid media, Microbispora rosea were able to degrade it by 50% (Tokiwa et al., 2009).

1.4.4 Polylactic acid

PLA is a renewable, aliphatic polyester that is made up of lactic acid (2-hydroxy propionic acid) that can be produced by fermentation of renewable resources like starch materials, cane, molasses and cellulose (Kumar and Maiti, 2016; Karamanlioglu et al., 2017). This polymer accounts for 24% of the global production capacity of biodegradable polymers (Haider et al., 2019). PLA is considered as a hydrophobic and semi-crystalline polymer that can be synthesized by conventional chemical engineering. It has a higher melting point (170°C) and a higher glass transition temperature (60°C) than the other aliphatic polyesters (Nakamura et al., 2001; Leja and Lewandowicz, 2010). However, above its melting point the chains of the amorphous region of PLA become flexible and hence it will be exposed to degradation and composting.

This bio-based product has many excellent properties compared to other aliphatic polyesters such as strength, stiffness, and gas permeability (Hamad et al., 2018). It has also high mechanical strength, high modulus, biodegradability, biocompatibility, bioabsorbability, transparency, energy savings, low toxicity and easy processability (Siracusa et al., 2008; Song et al., 2009; Qi et al., 2017). The chemical structure of PLA is

([-O(CH3)CHCO-]n) (Tokiwa et al., 2009).

PLA shows potential applications in so many fields. It has been widely used in the biomedical and pharmaceutical fields due to its biocompatibility and biodegradability in

32 contact with living bodies (Avérous, 2013). Moreover, PLA has many domestic applications such as bottles, cups, apparel, food packaging, children toys and many other domestic applications. In addition, this polymer has engineering and agricultural applications (Avérous, 2013; Sin et al., 2013).

Because of the presence of biotic and non-biotic factors, the degradation of PLA can be influenced by many processes: chemical, physical and biological processes and hence different degradation mechanisms can be involved with the degradation of the polymer (Nampoothiri et al., 2010). However, regarding biological degradation, PLA can be fully degradable when composted with temperatures of 60°C and above. The degradation starts via hydrolysis to water-soluble compounds and lactic acid and later these compounds will be broken down by different microorganisms into CO2, water and biomass (Pranamuda and Tokiwa, 1999; Shah et al., 2008).

1.5 Biological degradation of biodegradable polymers

Any change that might alter the physical or chemical properties of the polymer to cause breakage of the bonds and subsequent chemical transformation is called polymer degradation. These changes can be a chemical, physical or biological reaction caused by some environmental factors such as light, heat and moisture (Shah et al., 2008; Song et al., 2009; Leja and Lewandowicz, 2010; Chinaglia et al., 2018). There are different types of polymer degradation: photo, thermal or biological (Krzan et al., 2006). Thermal degradation is the degradation that uses high amount of heat, more than the ambient temperature. This degradation will lead in physical and optical changes and it will change the molecular weight of the polymer as well (Krzan et al., 2006; Kumar and Maiti, 2016). Photodegradation on the other hand uses high frequency electromagnetic radiation such as UV light and gamma rays to degrade the polymer which will cause chain scission in polymeric molecules and therefore a change in the molecular weight which subsequently allow it to biodegrade (Krzan et al., 2006; Shah et al., 2008; Kumar and Maiti, 2016). Biological degradation (biodegradation) is the mineralisation of polymeric materials (organic materials) via the action of naturally occurring microorganisms such as fungi and bacteria and their enzymes (enzymatic degradation) (Gómez and Michel, 2013). The degradation of organic materials can take place either aerobically (in the presence of oxygen) or anaerobically (without oxygen) (Shah et al., 2008; Leja and Lewandowicz, 2010; Gironi and Piemonte, 2011). The aerobic degradation is more efficient than 33 anaerobic degradation because the former produces more energy due to the presence of

O2 (Pathak and Navneet, 2017). The rate of biodegradation is influenced by three factors: temperature, humidity, and type of microbes (Siracusa et al., 2008).

The process of biological degradation can take place in several steps as follows (Lucas et al., 2008; Kumar and Maiti, 2016; Bano et al., 2017; Haider et al., 2019):

1. The formation of biofilms, which happens by the adherence of the microorganisms to the polymer surface. This can be also called ‘micro-fouling’. The actions of microbial communities in combined with abiotic factors fragment the biodegradable materials into tiny fractions (Biodeterioration). 2. The microbial enzymes will break down the polymer into simpler forms such as dimers or oligomers (Depolymerization). 3. The uptake of these simpler molecules by microorganisms (Assimilation).

4. Mineralisation as the last step which is the production of metabolites like CO2, H2O

and CH4.

Another type of the biological degradation besides the microbial and the enzymatic degradation is composting. Composting is a natural, aerobic process where biodegradable materials such as plant biomass and manure or any other organic solid waste materials are decomposed and transformed into a humus-like substance called compost. This is a microbially rich process that results in the production of CO2, H2O, minerals, and stabilised organic matter in a controlled biological manner (Gironi and Piemonte, 2011; Zafar et al., 2013; Kumar and Maiti, 2016; Qi et al., 2017).

The composting process typically takes place in 12 weeks under elevated temperatures that may reach 65°C and is started by biotic and abiotic processes to degrade a polymer into low-molecular weight fragments. Later, these fragments will be used by the microorganisms (Mohanty et al., 2000; Gironi and Piemonte, 2011; Rujnić-Sokele and Pilipović, 2017). However, there are some quality criteria that the polymer product must meet to be recognised as compostable polymer and as well to avoid environmental and health consequences: (Mohanty et al., 2000; Song et al., 2009; Gironi and Piemonte, 2011; Rujnić-Sokele and Pilipović, 2017):

1. The product should not exceed several heavy metal limits. 2. The products should biodegrade within 6 months under controlled composting conditions and it should biodegrade by at least 90%. 3. The product should lack polymer residues; it should fragment sufficiently to visually undetectable components (<2 mm). 4. The product should lack ecotoxicity; it should not pose any negative effects to the germination and growth of plants.

On the other hand, there are many factors that can affect the biodegradation of the polymers these are (Tokiwa et al., 2009; Luyt and Malik, 2019):

1- The polymer properties: chemical and physical properties can have an impact on the biodegradability of the polymer. 2- The surface conditions of the polymer: like for example surface area, hydrophilic and hydrophobic properties. 3- The first order structure of the polymer: chemical structure, molecular weight and molecular weight distribution. 4- The high order structure of the polymer: for example glass transition temperature, melting temperature, modulus of elasticity, crystallinity and crystal structure.

Moreover, biodegradation depends on other factors like microbial activity of the environment (Reddy et al., 2003; Bano et al., 2017). All of these factors have a major impact on the biodegradation process of the polymers. For example, the molecular weight can influence the biodegradation process because it determines many physical properties. The degradability will decrease with the increase of the molecular weight

(Tokiwa et al., 2009). It has been shown that high molecular weight (Mn > 4000) PCL displays slower degradation than PCL with low Mn by Rhizopus delemar lipase (Tokiwa et al., 2009). Furthermore, the degree of crystallinity has strong impact on the biodegradation of the polymer and this is because the enzymes attack the amorphous domains of the polymer. In the amorphous domain the molecules are loosely packed, therefore easy to be degrade (Tokiwa et al., 2009). As the crystallinity of PLA increases, the rate of the degradation decreases (Tokiwa et al., 2009). In addition, higher melting temperature can decrease the rate of biodegradation of the polymer. Low molecular weight L-PLA and their copolymers can be hydrolysed by lipase but D-PLA and higher

35 molecular weight L-PLA cannot be hydrolysed easily because of their high molecular weight and high melting temperature (Kumar and Maiti, 2016).

1.6 Advantages and applications of biodegradable polymers

Biodegradable polymers are positive for many environmental issues because they can reduce the GHG emissions since they are synthesized from renewable feedstock. For example, PHA and lactic acid can be produced by microorganisms and agricultural products (Leja and Lewandowicz, 2010). Moreover, biodegradable plastics can also enhance the soil fertility and decrease the accumulation of synthetic plastic in the environment. Hence this will decrease the harm caused to wildlife through plastic ingestion and toxicity, and will reduce the economic costs for removing plastic waste in the environment ( Leja and Lewandowicz, 2010; Emadian et al., 2017). Furthermore, these polymers can be recycled into beneficial products (monomers and oligomers). This recycling can take place by microorganisms and enzymes (Leja and Lewandowicz,2010; Emadian et al., 2017). Therefore, new considerations of waste management strategies are now possible through the development of BDPs because these materials have been produced to degrade under environmental conditions or in municipal and industrial biological waste treatment facilities (Shah et al., 2008; Emadian et al., 2017).

Biodegradable polymers can be used instead of conventional polymers for modified atmospheric storage (MAP) of fruits and vegetables in which MAP can extend the storage life of many crops (Mangaraj and Goswami, 2011; Mangaraj et al., 2018). In this technology generating an atmosphere with low O2 and /or high in CO2 to influence the metabolism of the packed product is needed. In addition, this technology can improve moisture retention, and this can have an influence on preserving the product quality (Mangaraj and Goswami, 2011; Mangaraj et al., 2018).

Moreover, the use of biodegradable and biorenewable products can cause less oil consumption and the CO2 produced from the incineration of this waste will be equivalent to the amount of CO2 fixed during photosynthesis by plants that provided the raw materials for the biopolymers and hence global warming through reduced GHG emission will be reduced. This concept is for carbon neutral or ‘zero emission’ bioplastic (Funabashi et al., 2009). For example, the PHB biopolymer has attracted the attention of the scientific community by its low CO2 emission characteristics (Mostafa et al., 2018).

There are extensive applications of BDPs in different domains, like medical and ecological (agricultural) and goods packaging. Just like for the synthetic plastics, there is dramatic increase in the use of BDPs packaging materials (Huang et al., 1990). This includes bags, wrappings, loose-fill foam, food containers, film wrapping, and laminated paper (Song et al., 2009). In the medical area, BDPs have been used in surgical implants in vascular and orthopaedic surgery, implantable matrices for the controlled, long-term release of drugs inside the body, absorbable surgical sutures, and for use in the eye (Huang et al., 1990). In addition, BDPs can be used as well in tissue engineering, gene therapy, regenerative medicine, temporary implantable devices (Doppalapudi et al., 2014). For example, PCL has been wildly used as scaffolds for tissue engineering due to its excellent biocompatibility (Nair and Laurencin, 2007). In the agricultural domain, these polymers can be used in mulch films and planters (Song et al., 2009). Table 1.2 shows some applications and uses for the most commonly used BDPs.

Table 1.2 Applications of some widely used biodegradable polymers.

Biopolymer Applications References Slow release systems for drugs, mulch and other agricultural films, fibers (Shah et al., 2008; Imre PCL containing herbicides to control aquatic and Pukánszky, 2013) weeds, seedling containers. Products like bottles, bags, wrapping (Shah et al., 2008; Babu et film, disposable nappies, materials for PHB al., 2013; Imre and tissue engineering, scaffolds and for Pukánszky, 2013) controlled drug release carriers Packaging, paper coatings, sustained (Shah et al., 2008; Babu et release systems for pesticides and al., 2013; Imre and PLA fertilizers, mulch films, and compost Pukánszky, 2013; Sin et al., bags. 2013) Packaging films, dishware, medical materials, bags, mulch film, flushable (Xu and Guo, 2010; Babu et PBS hygiene, food container, textiles and al., 2013) disposable medical devices Polyglycolic controlled drug releases, implantable (Shah et al., 2008) acid (PGA) composites and bone fixation parts Orthopedic implant devices as bone (Babu et al., 2013; Imre Starch fillers, Food applications and Bone and Pukánszky, 2013) cements.

1.7 Environmental impact of the degradation process on biota and its quantifications

BDPs can be broken down in the environment by the action of various factors such as temperature, humidity, pH, sunlight, alongside the action of microorganisms and their enzymes (Song et al., 2009; Krasowska et al., 2016; Emadian et al., 2017). The degradation of these BDPs is suggested to be environmentally acceptable since it should not lead to the production of toxic or harmful materials (Krasowska et al., 2016). Soil microorganisms

38 can be an indicator of the environmental conditions because they are sensitive to the environmental changes due to many factors such as pollution (Adhikari et al., 2016). This study is focused on fungi which are known to have an important role in the degradation process of BDPs as demonstrated by many studies (Rutkowska et al., 2002; Tokiwa et al., 2009; Emadian et al., 2017).

Fungi play an important and a principal role in the ecosystem. They can be involved in litter degradation as well as their capability in producing extracellular enzymes which can attack some BDPs bonds (Ma et al., 2013). They are important pathogens of plants and animals as well as mycorrhizal symbionts of plants (Anderson et al., 2003). Moreover, fungi are considered as a source of nutrients and as primary recyclers of dead matter (Wooley et al., 2010). Therefore it is very important to study any change in microbial communities because any variations in the microbial community structure can influence ecosystem processes (Agrawal et al., 2015).

The sequencing of DNA extracted from environmental samples offers a unique opportunity to study whole microbial communities. These sequences can be approached by the use of next generation sequencing (NGS) technology (De Beeck et al., 2014). NGS is a sequencing method that can perform huge parallel sequencing in which massive numbers of fragments of DNA are sequenced from a single sample (Grada and Weinbrecht, 2013). The use of NGS for the analysis of complex microbial communities has increased recently because this method has low cost and can provide higher throughput alternative to sequencing DNA compared to traditional Sanger sequencing and traditional culture based methods (Grada and Weinbrecht, 2013; Clooney et al., 2016). NGS is beneficial in many ways like for example, determining the role of the microbiome obtained from environmental samples like soil and compost, or to study the diversity and abundance of whole microbial communities (Clooney et al., 2016) as well as in clinical diagnostics, (Grada and Weinbrecht, 2013) and the analysis of important ecosystem functional genes (Lemos et al., 2017).

However, there are a few limitations and challenges for NGS technology like for example the inaccuracy in sequencing homopolymer regions (which are the spans of repeated nucleotides) on certain NGS platforms. In addition, the data analysis is considered as time consuming and a challenging issue on how to convert an immense amount of genetic data

39 into rational biological conclusions. This requires special background of bioinformatics (Grada and Weinbrecht, 2013; Lemos et al., 2017).

For using this technique in this study, Illumina (MiSeq) platform has been used to sequence the ITS region, which is between the 5.8S and 26S rRNA genes of the fungal communities present in the compost. Internal transcribed spacer (ITS) of fungal ribosomal DNA is a piece of nonfunctional RNA that can be found between structural ribosomal RNAs and composed of two subregions , ITS1 and ITS2 (Hao et al., 2013). This region is considered to have the highest chance for successful identification of an extensive range of fungi (do Nascimento Barbosa et al., 2016). However, only part of this region can be used -ITS2 subregion used in this study- due to the read length limitations of illumina sequencing (Yang et al., 2018).

1.8 Thesis aims and objectives

The overall aim of this research is to determine the characteristics of degradation of PCL biodegradable polymer and compare it with the degradation of other BDPs in terms of the time required for the degradation rate of these BDPs under controlled and environmental conditions. This study also aims to investigate the degradation of PCL in different forms (strips and powder) and whether the degradation of this polymer in the environment has an effect on the fungal community structure in compost and the germination and growth of seeds. The aims of this thesis are addressed in four chapters as follows:

1. To determine the rate of microbial degradation of four biodegradable polymers in soil and compost under different conditions.

This aim is addressed in Chapter 2 ‘A comparative study on the rate of microbial degradation of four biodegradable polymers in soil and compost’. In this chapter the rate of degradation of four biodegradable polymers: PCL, PHB, PBS and PLA were compared in both soil and compost under controlled conditions and the principal colonising fungi were identified. The degradation rate was also compared under environmental conditions.

2. To investigate the degradation of PCL polymer in different forms to determine the characterisation of PCL as a promising biodegradable polymer.

This aim is addressed in Chapter 3 ‘Characterisation of polycaprolactone as a promising biodegradable polymer’. In this chapter different forms of PCL polymer (strips and powder) have been studied for their degradation rate and compared with the degradation rate of the PCL in disc form (from Chapter 2) to help determine the characterisation of PCL.

3. To determine the impact of PCL degradation on microbial communities in compost at different temperatures using next generation sequencing.

This aim is addressed in Chapter 4 ‘The impact of polycaprolactone degradation on microbial communities in compost at different temperatures using next generation sequencing’. Towards this aim, NGS technique is used to quantify fungal communities structures in 10% PCL in compost samples at five different temperatures and compared them with fungal communities structures in the initial compost (without PCL). 41

4. To determine if PCL degradation has any effect on seed germination in compost.

This aim is addressed in Chapter 5 ‘The effect of polycaprolactone degradation in compost on seed germination’. This chapter investigated the effect of PCL degradation in compost on seed germination at different PCL concentrations which have been mixed with compost and incubated at different temperatures for eight weeks to emulate the natural environment and the different stages of composting process. The extractions of these compost/PCL mixtures have been used for seed germination.

1.9 References

Adamopoulou, E. (2012) Poly(butylene succinate) : A Promising Biopolymer (Master's thesis). Athens University.

Adhikari, D., Mukai, M., Kubota, K., Kai, T., Kaneko, N., Araki, K. S. and Kubo, M. (2016) ‘Degradation of Bioplastics in Soil and Their Degradation Effects on Environmental Microorganisms.’ Journal of Agricultural Chemistry and Environment, 5(1) pp. 23–34.

Agrawal, P. K., Agrawal, S. and Shrivastava, R. (2015) ‘Modern molecular approaches for analyzing microbial diversity from mushroom compost ecosystem.’ 3 Biotech, 5(6) pp. 853–866.

Ali, F. B. and Mohan, R. (2009) ‘Thermal, mechanical, and rheological properties of biodegradable polybutylene succinate/carbon nanotubes nanocomposites.’ Polymer Composites, 31(8), pp.1309-1314.

Anderson, I. C., Campbell, C. D. and Prosser, J. I. (2003) ‘Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil.’ Environmental Microbiology, 5(1) pp. 36–47.

Andrady, A. L. and Neal, M. a (2009) ‘Applications and societal benefits of plastics.’ Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 364(1526) pp. 1977–1984.

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Chapter 2

A comparative study on the rate of microbial degradation of four biodegradable polymers in soil and compost

2.1 Abstract

Plastics are an indispensable and basic material in all aspects of the modern world. The durability and diverse properties of plastics make them ideal materials in the manufacture of a wide range of products. However, the recalcitrant nature of many plastics means that they are problematic in terms of disposal and are a major industrial waste product and environmental pollutant. The use of biodegradable polymers can aid in resolving a number of waste management issues as they are degraded ultimately to CO2 and water and have the potential to be directed to conventional industrial composting systems. Four different biodegradable polymers (BDPs), polycaprolactone (PCL), polyhydroxybutytate (PHB), polylactic acid (PLA) and polybutylene succinate (PBS) were used to study the time required for biodegradation to occur in soil and compost under controlled and environmental conditions. Degradation of polymer discs was measured by monitoring changes in disc weight over a period of more than ten months under controlled conditions at three different temperatures: 25°C, 37°C and 50°C. Polymer discs were also monitored for its weight change when buried in soil under environmental conditions over a period of 21 months. Degradation rates varied between the polymers and the incubation temperatures. PCL showed the fastest degradation rate under controlled and environmental conditions and was completely degraded when buried in compost and incubated at 50°C after 91 days. Microorganisms present on the surface of the polymers under controlled conditions were isolated and identified. Thermomyces lanuginosus was found to be the main degrader of PCL at 50°C when incubated in compost.

2.2 Introduction

Over the last six decades, the use of plastic materials has had a major impact on society and has become essential due to their extensive and diverse range of applications. The global production of plastics has increased every year (Tokiwa et al., 2009). There are wide spread applications and usage of plastics because of their favourable mechanical and thermal properties and because plastics are cheap to manufacture, stable and durable. The annual production of petroleum based plastics exceeded 300 million tonnes in 2015 (Emadian et al., 2017).

Despite the wide range of applications and benefits of plastics, the recalcitrant nature of many plastics means that they stay in the environment for decades or even centuries. As a consequence, significant amounts of plastic polymers accumulate in the environment and landfills resulting in environmental pollution and waste management issues (Hopewell et al., 2009). Therefore and due to the growing environmental problems associated with plastic accumulation, BDPs are considered as one potential solution (Zheng et al., 2005; Tokiwa et al., 2009; Haider et al., 2019).

BDPs are polymeric materials that can be decomposed into carbon dioxide, methane, water, inorganic compounds or biomass by the enzymes of microorganisms (Song et al., 2009; Laycock et al., 2017; Haider et al., 2019). The chains of BDPs can also be broken down by non-enzymatic processes, such as chemical hydrolysis (Tokiwa et al., 2009).

Processing of atmospheric CO2 by plants can produce BDPs and thus these polymers can be naturally recycled by biological processes without impacting on atmospheric CO2 levels (Tokiwa et al., 2009).

A number of different types of biodegradable polymers have been developed to date and several studies have investigated the degradability of these plastics. However, relatively little is known about the time required for these materials to be fully degraded in soil and compost. Moreover, most studies have concentrated on monitoring degradation of polymer powders or as polymer blends (biodegradable polymers mixed with starch or fibres or any other degradable materials). In this study, the rate of degradation of four BDPs, PHB, PCL, PBS and PLA were compared under both controlled and environmental conditions. The principal colonising fungi were isolated and identified.

2.3 Materials and methods

2.3.1 Plastic materials

PHB granules (5mm; ALDRICH chemistry), PCL granules (Mn 80,000; ALDRICH chemistry), PBS granules (Goodfellow Cambridge Limited), and PLA granules (5mm; Goodfellow Cambridge Limited) were used. Plastic sheets were prepared by melting the polymer pellets in a halogen oven for 20 min at the following temperatures: 180°C for PLA, 80°C for PCL, 195°C for PHB and 210°C for PBS. For soil/compost burial studies, plastic discs were cut from the sheets into two sizes ca. 1 cm diameter and 3 mm thick for the controlled conditions investigations and ca. 5 cm diameter and 2.5 mm thick for the environmental burial (uncontrolled conditions) investigations.

2.3.2 Soil and compost

Commercial soil and compost were obtained from the Compost shop (UK) and screened using a 7 mm sieve prior to use. The percentage moisture content was calculated by drying a known weight of soil or compost (Wini) in triplicate at 55°C until it reached a constant dry weight (Wfinal). The percentage moisture content was calculated using the following equation:

Percentage moisture content = (Wini –Wfinal) / (Wini) x 100

Wini = Initial weight (g)

Wfinal = Final weight (g)

The water holding capacity (WHC) for the soil and compost was determined by weighing 25 g of soil/compost that was then put into a funnel lined with filter paper and connected to a graduated cylinder. A 50 ml volume of water then was added to soil/compost in the funnel and then allowed to drain and collect in the graduated cylinder. The percentage water holding capacity was then calculated using the following equation:

(Volume of water added – volume of water retained)/ (initial weight of soil) x100

2.3.3 Biodegradation of polymer discs under controlled conditions

In order to investigate the biodegradation of the polymers under controlled conditions, rectangular 1 L plastic boxes (Sealfresh, Stewart, UK) with dimensions 16.5 cm x 11.5 cm x 5 cm were filled with 500 ml of soil or compost and seven pre-weighed polymer discs

53 were buried vertically ca. 2 cm below the surface. Plastic boxes were sealed with lids in which three holes had been made (ca. 1 cm diameter) to allow for gaseous exchange and covered with a single layer of parafilm. Boxes were incubated at 25° or 37°C (soil) or 25°, 37° or 50°C (compost) for 10 months. Boxes were weighed every week to check for water loss through evaporation and replaced with sterile water using a plant spray. To determine changes in the weight of the polymer discs, discs were weighed periodically after the removal of loosely bound soil/compost with a soft brush and weight loss was determined according the following equation:

Weight loss (%) = ( Wdry / Wini) x 100

Where Wini is the initial weight of the disc before burial and Wdry is the dry weight of disc after burial. Polymer discs under study were also measured for changes in thickness and surface area during burial using a digital caliper 0-150 mm (data not shown).

2.3.4 Biodegradation of polymer discs under environmental conditions

In order to investigate the biodegradation of the polymers under environmental conditions, polymer discs were buried in soil in an elevated bed in a field environment (Michael Smith building quad, The University of Manchester). The samples were randomly located in the bed and three replicates from each polymer were sampled every two months for PCL, PBS and PHB and every four months for PLA over a period up to 21 months. Unburied control discs were kept in a dry indoor environment and were weighed periodically to make sure that there was no weight change. Another set of control discs were put in sterile water over the 21 months and their weight was measured at the end of the period to investigate if there was any effect of water on the degradation of the polymers.

2.3.5 Isolation and identification of fungal growth on the surface of the polymers under controlled conditions

Fungi were recovered from the surface of the polymer discs and this recovery was adapted using a previously described methods (Cosgrove et al., 2007; Karamanlioglu et al., 2014). Polymer discs were recovered randomly from the compost and soil at all temperatures and placed in 1 ml phosphate-buffered saline. The surfaces of the discs were then scraped 3 times on both sides using a sterile razor blade. Biomass suspension obtained from the surface of the polymer discs was plated onto Potato Dextrose agar 54

(PDA) plates supplemented with chloramphenicol (50 µg/ml) after serial dilution and incubated at temperatures from where the discs were obtained.

2.3.6 Genomic DNA extraction from fungal mycelia

Distinct colonies from PDA plates were purified by sub culturing onto PDA plates and incubated at their designated temperatures and then used for DNA extraction according to Feng et al. (2010). From each plate, a loopfull of mycelium or spores were added to a 2 ml tube containing glass beads (0.5 mm diameter). 0.7 ml of lysis buffer (100mM Tris-HCL, pH 8.0; 50 mM EDTA, pH 8.0; 1% (v/v) SDS) and 10 µl of RNaseA (10 µg/ml) were added to the tube and vortexed for 5 s and then homogenised twice for 30 s at 5000 rpm using a Bead Bug Microtube Homogenizer (Merck, UK). Following centrifugation at 13,000 rpm, the supernatant was transferred into a new tube and 100µl of potassium acetate buffer (3.0 M, pH 5.5) added and centrifuged at 13,000 rpm for 2 min. 0.5 ml of the supernatant was transferred into 1.5 ml tube containing isopropanol and centrifuged at 13,000 rpm for 2 min. Supernatant was discarded and the DNA pellet was washed with 0.8 ml 80% (v/v) ethanol and centrifuged at 13,000 rpm for 30 s and then the ethanol was removed. DNA was air-dried and dissolved in 50 µl DEPC (diethylpyrocarbonate) water. DNA then was quantified using a Nanodrop™ 1000 machine with ND-1000 3.1.0 software (Thermo Fisher Scientific Inc, USA) and stored at -20°C.

2.3.7 DNA amplification of fungal isolates

In order to identify the fungal isolates, the ITS1-5.8S-ITS2 rRNA gene complex was amplified using universal fungal primers (White et al., 1990)

ITS1-F (5´-CTTGGTCATTTAGAGGAAGTAA-3´) and

ITS4-R (5´-TCCTCCGCTTATTGATATGC-3´). The Polymerase Chain Reaction (PCR) mixture contained 25 µl MyTaq™ Red Mix (Bioline reagent limited, UK), 1 µl (100x) Bovine Serum Albumin (BSA), 5 µl of extracted DNA (75- 100 ng/ µl), 2 µl of each primer (10µM) and the total volume made up to 50 µl with DEPC water. The PCR cycle conditions were as follows: 35 cycles with 95°C denaturation for 1 min, annealing at 56°C for 15 s and extension at 72°C for 10 s. The PCR products were separated on a 1.0 % (w/v) agarose gel (Sigma, Aldrich, Germany) with a DNA hyperladder™ 1kb (Bioline reagent limited, UK) and visualised using UV light machine (UVitec). 55

2.3.8 Purification, sequencing and identification

The QIAquick PCR purification kit (Qiagen Ltd, UK) was used to purify the PCR products according to the manufacturer’s instructions. Later 10 ng of purified PCR products were mixed with 4 pmoles of a single relevant primer and the volume of each reaction was made up to 10 µl with DEPC water and sent for sequencing facility at The University of Manchester (DNA sequencing facility). The DNA sequences were then viewed using FinchTV.Inc. The sequences were used to interrogate the National Centre for Biotechnology Information (NCBI) database using BLAST (Basic Local Alignment Search Tool). Isolates recovered from the surface of the polymers were identified by choosing the closely related species on NCBI nucleotide database.

2.3.9 Degradation ability of fungal isolates on PCL strips

The capability of fungal isolates that recovered from the surface of PCL at 50°C in compost to degrade PCL polymer (as the fastest degraded polymer) were determined. PCL were made into strips by melting 12.5 g of PCL pellets in 100 ml dichloromethane solvent. The produced sheet was then cut into strips (6 x 0.5 cm) to be used for the experiment. More details about the preparation of PCL strips are discussed in the following chapter (chapter three). Modified methods from Cosgrove et al. (2010) and Crabbe et al. (1994) were used. 200 g of wheat grain was used as a matrix for fungal growth. Wheat grain was put in a wide glass container with a lid and autoclaved. 150 ml of a yeast-extract salts medium (YES) was added to the wheat grains. Two fungal isolates PCL(A) and PCL(B), that were grown on PDA media for one week were chopped and added to the sterile wheat individually (each strain was added to a separate container). One container was left uninoculated as a control. The containers were then incubated at 50°C (the temperature from which the strains were isolated) for one week before the addition of the PCL strips to allow the culture to grow. After one week, PCL strips were added after surface sterilisation with 70 % (v/v) ethanol.

2.3.10 Tensile strength measurement

PCL strips of 0.5 cm x 6 cm size were used to assess the degradation of PCL polymer by the isolated fungi from 50°C using tensile strength measurements. The strips were recovered every three days and the tensile strength measured using a T-series Tensile Test Machine (Tinius Olsen, LTD. Surrey, UK) supported with QMAT Professional software.

2.3.11 Scanning Electron Microscopy

Polymer discs recovered from compost and soil under controlled conditions at 25°C, 37°C and 50°C at random time points were observed for fungal growth by Scanning electron microscopy (SEM) (FEI, QUANTA FEG 250, Netherlands). Air dried samples were mounted for sputter coating with gold/palladium on a Quorum SC7620 sputter coater. The samples were then imaged at high vacuum in an FEI Quanta 250 FEG at 10kV.

2.3.12 Statistical analysis

Analysis of variance (One Way ANOVA) test on SPSS were used to estimate any significant change in the weight of the discs over time with the significance P value <0.05.

2.4 Results

2.4.1 Soil and compost analysis

The moisture content of the compost and soil were found to be 35% and 26%, respectively, and the water holding capacity was 75% and 60%, respectively. The pH was 7.1 and 7.0 for compost and soil, respectively.

2.4.2 Weight change of polymer discs buried under controlled conditions

To determine the weight change of polymer materials, polymer discs were recovered every week from compost and every three weeks from soil that were incubated at 25°C, 37°C and 50°C (compost only) and the percentage of the original disc weight were calculated (Figures 2.1 to 2.4). The greatest degree of degradation occurred with PCL where total weight loss occurred in compost at 50°C after 91 days (Figure 2.1A). The half- life of the PCL discs (the day when the disc weight was reduced by 50%) was estimated to be 45 days. While a significant (P<0.05) reduction in weight was also observed at 25°C and 37°C in both soil and compost, the reduction was less and slower compared to the 50°C treatment and therefore temperature was a major factor in PCL degradation. PHB showed a significant (P<0.05) reduction in weight under all conditions although less than the reduction of PCL polymer except at 50°C in compost. Weight loss in PHB discs at 50°C in compost decreased rapidly after 150 days (Figure 2.2). While no significant change (P>0.05) was observed for PLA at 25°C and 37°C in both soil and compost, a significant (P<0.05) reduction was observed at 50°C toward the middle of the burial period until complete degradation was recorded, as shown in Figure 2.3A. 57

From the data in Figure 2.4, it is apparent that there was moderate change in the weight of PBS discs at 50°C in compost and 37°C in soil, which showed significant reduction (P<0.05). However, no significant (P>0.05) reduction at 25°C and 37°C in compost and at 25°C in soil was detected.

A 120

25°C

100

37°C 80 50°C

40 % originalofweight %

0 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300

Time (Days)

B 120

100

25°C

80 37°C

% of original weight originalof % 20

0 0 50 100 150 200 250 300

Time (Days)

Figure 2.1 Percentage weight change over time of PCL discs buried in compost or soil. PCL discs were buried in either compost (a) or soil (b) in a laboratory microcosm and incubated at 25°C, 37°C or 50°C (compost only) and the mean weight change calculated at ca. 7 day intervals for up to 270 days. Each point represents the average of percentage weight of seven replicates. The error bars represent the standard error of mean.

120 A

100

25°C 80 37°C 50°C

40 % originalofweight %

0 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300

Time (Days)

B 120 25°C

100 37°C

40 % of original weight originalof % 20

0 0 50 100 150 200 250 300 Time (Days)

Figure 2.2 Percentage weight change over time of PHB discs buried in compost or soil. PHB discs were buried in either compost (a) or soil (b) in a laboratory microcosm and incubated at 25°C, 37°C or 50°C (compost only) and the mean weight change calculated at ca. 7 day intervals for up to 270 days. Each point represents the average of percentage weight of seven replicates. The error bars represent the standard error of mean.

A 120

100

25°C 60 37°C 50°C

% originalofweight % 40

0 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300

Time (Days)

B 120

100

60 25°C 37°C 40

% originalofweight % 20

0 0 50 100 150 200 250 300

Time (Days)

Figure 2.3 Percentage weight change over time of PLA discs buried in compost or soil. PLA discs were buried in either compost (a) or soil (b) in a laboratory microcosm and incubated at 25°C, 37°C or 50°C (compost only) and the mean weight change calculated at ca. 7 day intervals for up to 270 days. Each point represents the average of percentage weight of seven replicates. The error bars represent the standard error of mean.

A 120

100

80 25°C 60 37°C 50°C

40 % originalofweight %

0 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300

Time (Days)

B 120

100

25°C 60 37°C

40 % of original weight originalof %

0 0 50 100 150 200 250 300 Time (Days)

Figure 2.4 Percentage weight change over time of PBS discs buried in compost or soil. PBS discs were buried in either compost (a) or soil (b) in a laboratory microcosm and incubated at 25°C, 37°C or 50°C (compost only) and the mean weight change calculated at ca. 7 day intervals for up to 270 days. Each point represents the average of percentage weight of seven replicates. The error bars represent the standard error of mean.

Table 2.1 Summary of percentage weight change of polymers in soil and compost at various temperatures over 270 days. Data represents the mean of 7 replicates ± SEM. * = significant (P<0.05) reduction (student t-test).

Temperature Mean % remaining weight

(°C) PCL PBS PLA PHB

25 20.4±3.3* 96.5±1.5 101.5±0.3 40.6±4.0*

Compost 37 20.5±9.2* 88.5±3.5 102.9±0.4 21.5±1.5*

50 0* 43.4±5.03* 0* 4.6±2.0*

25 43.9±3.8* 92.6±2.5 105.2±0.7 77.6±2.4* Soil 37 48.3±2.7* 56.9±3.9* 105.2±0.8 42.2±3.4*

2.4.3 Degradation of polymers buried in soil under environmental conditions

In order to compare the rate of degradation of the four polymers under natural environmental conditions, discs buried outside in soil were weighed over a time period of 21 months. Control discs of all polymers were incubated in two conditions: dry conditions at room temperature and in sterile water at room temperature. Both controls were in triplicate for each polymer type.

The results showed that while there was a significant difference (P< 0.05) in the weight of PCL polymer before and after burial, there was no significant weight loss for PHB, PBS and PLA ( P>0.05). Figures 2.5 – 2.8 show the change in weight of PCL, PHB, PBS and PLA before and after burial.

initial wt Final wt 4.50 4.00 3.50 3.00 2.50

2.00 Mean weight (g) 1.50 1.00 0.50 0.00 0 1 2 3 4 5 6 7 8 9 10 water Time (Months) control

Figure 2.5 Mean of weight over time of PCL polymer buried in soil under environmental conditions. Each bar represents the average weight of three replicates as the initial weight (the weight before the burial of the discs) and the final weight (the weight after disc recovery). The error bars represent the standard error of mean.

Initial wt 5.0 Final wt 4.5 4.0

3.5 3.0 2.5 2.0

Mean weight (g) 1.5 1.0 0.5 0.0 0 1 2 3 4 5 6 7 8 9 10 water Time (Months) control

Figure 2.6 Mean of weight over time of PHB polymer buried in soil under environmental conditions. Each bar represents the average weight of three replicates as the initial weight (the weight before the burial of the discs) and the final weight (the weight after disc recovery). The error bars represent the standard error of mean.

Initial wt 5.0 Final wt

4.5

4.0 3.5 3.0 2.5

2.0 Meanof weight (g) 1.5 1.0 0.5 0.0 0 1 2 3 4 5 6 7 8 9 10 water control Time (Months)

Figure 2.7 Mean of weight over time of PBS polymer buried in soil under environmental conditions. Each bar represents the average weight of three replicates as the initial weight (the weight before the burial of the discs) and the final weight (the weight after disc recovery). The error bars represent the standard error of mean.

Initial wt Final wt 5.0 4.5

4.0 3.5 3.0 2.5 2.0

Meanof weight (g) 1.5 1.0 0.5 0.0 0 1 2 3 4 5 water control Time (Months)

Figure 2.8 Mean of weight over time of PLA polymer buried in soil under environmental conditions. Each bar represents the average weight of three replicates as the initial weight (the weight before the burial of the discs) and the final weight (the weight after disc recovery). The error bars represent the standard error of mean.

2.4.4 Changes in physical appearance of the polymers buried in soil under environmental conditions

The changes in the physical appearance of the polymers that have been buried in soil under environmental conditions were examined visually. The fungal growth can be seen very clearly on the surface of PCL discs (Figure 2.9A) and PBS discs (Figure 2.9C). PLA discs showed the least changes among all other polymers (Figure 2.9D).

A B

C D

Figure 2.9 Changes in physical appearance of polymer discs with time under environmental conditions for (A) PCL, (B) PHB, (C ) PBS and (D) PLA. The first disc in each panel represent the control disc at dry indoor environment and the following discs represents the degradation change every two months for the total of 21 months for all polymers except PLA which their discs represents the degradation change every four months. The last disc in each panel represents the water analysis disc.

2.4.5 Identification of polymer degrading fungi recovered from the surface of the polymers buried under controlled conditions

Random discs were chosen from each pot at each temperature for the isolation of the potential polymer degrading fungi. The discs were chosen according to their appearance and how much they were covered by fungi before they were fully degraded. After fungal isolation, distinct morphotypes were identified and recovered from the surface of each polymer at different temperatures. The number of morphotypes growing on PDA plates following isolation from PCL and PHB at different temperatures are shown in Table 2.2 and 2.3.

PCL showed a higher number of morphotypes under compost and soil at 25°C and 37°C compared with PHB. However, Both PCL and PHB got the same number of morphotypes in compost conditions at 50°C. On the other hand, The PLA polymer showed three distinct morphotypes under 50°C only and PBS showed a single mprphotype under 50°C in compost and 37°C in soil (data not shown in the Table 2.2 and 2.3).

Table 2.2 Number of morphotypes growing on PDA plates isolated from the surface of PCL at different temperatures.

Temperature Compost Soil 25°C 3 1 37°C 2 2 50°C 2 *

* There were no polymer discs incubated at 50°C under soil condition.

Table 2.3 Number of morphotypes growing on PDA plates isolated from the surface of PHB at different temperatures.

Temperature Compost Soil 25°C 2 No growth 37°C 1 1 50°C 2 *

* There were no polymer discs incubated at 50°C under soil condition.

In order to identify the fungal isolates, genomic DNA was extracted and used as template for PCR using ITS1-ITS2 primers. Most of the thermophilic isolates recovered from the surface of the polymers PCL, PLA and PHB buried at 50°C in compost were identified as Thermomyces lanuginosus (Table 2.4). However, isolates recovered from the surface of PBS showed 99% sequence identity to Acremonium cellulolyticus, Penicillium pinophilum and Talaromyces verruculosus.

The most frequently recovered isolates from the surface of all polymers, with the exception of PLA, buried at 25°C and 37°C in both compost and soil was Aspergillus fumigatus (Table 2.4). The isolates that were homologues to Fusarium sp. were also recovered from PCL and PHB at 25°C in both compost and soil.

Another isolate that was recovered from PLA and PHB at 50°C in compost was 100% identical to Sordariales sp. Sequence. Furthermore, a strain that was homologous to Neocosmospora sp. was found on the surface of PCL at 25°C in compost (Table 2.4).

Table 2.4 Identification of the putative polymer degraders isolated from the surface of the polymers buried in soil and compost under controlled conditions.

Isolate Isolate Identity% Isolate NCBI identification temperature condition (Accession no.) PCL (A) 99 (KT365229.1) 50°C compost PCL (B) Thermomyces lanuginosus 98 (KY848520.1) PCL (C) 99 (KT365229.1) PLA ( E ) 97 (KT365229.1) 50°C compost Thermomyces lanuginosus PLA ( F) 99 (KT365229.1) 50°C compost PLA (G) Sordariales sp 100 (JN659504.1) 50°C compost PHB (H) Thermomyces lanuginosus 99 (KT365229.1) Sordariales sp 99 (JN659492.1) 50°C compost PHB ( I ) Scytalidium thermophilum 99 (AB085928.1) Chaetomium thermophilum 99 (AB746179.1) Talaromyces pinophilus 99 (MF686817.1) 50°C compost PBS (K) Acremonium cellulolyticus 99 (AB474749.2) Penicillium pinophilum 99 (AB474749.2) 37°C compost PHB (J) Aspergillus fumigatus 99 (KR527135.1) PCL (N) 99 (KX090325.1) 37°C compost Aspergillus fumigatus PCL (O) 98 (KX090348.1) 37°C soil PBS (L) Aspergillus fumigatus 100(KF494830.1) 37°C soil PHB (M) Aspergillus fumigatus 99 (KP724998.1) PCL (P) 100 (KY450779.1) 37°C soil Aspergillus fumigatus PCL( X) 99 (KR527135.1)

25°C compost PCL (Q) Neocosmospora ramosa 99 (KY031973.1)

25°C compost PCL ( R) Fusarium solani 99 (KX929306.1)

25°C compost PCL ( S ) Aspergillus fumigatus 99 (KP724998.1) 25°C compost PHB (V) Fusarium solani 99 (KX929306.1)

25°C compost PHB (W) Aspergillus fumigatus 100 (KX090348.1) 25°C soil PCL (T) Aspergillus fumigatus 100 (KR527135.1) 25°C soil PCL (U) Fusarium solani 99 (KM268689.1)

2.4.6 Biodegradability test

Further biodegradation analysis was performed on the PCL polymer. In order to investigate the ability of the isolated fungal strains to degrade PCL, two of the strains that were isolated from the surface of PCL in compost at 50°C were tested for their ability to degrade PCL strips using tensile strength measurements. These strains were PCL (A) and PCL (B), which were 99% and 98% identical, respectively to Thermomyces lanuginosus sequence. Degradation of PCL strips was assessed over two weeks by tensile strength measurement every three days and control strips not treated with fungi were measured at the same time points

Control strips showed insignificant change in tensile strength (P > 0.05). However, tensile strength of PCL strips incubated in the sterile wheat inoculated with the PCL (B) strain dropped drastically with a significant loss (P < 0.05) in tensile strength after 72 hours and this degradation was faster compared to the degradation of PCL strips incubated with the PCL (A) strain (Figure 2.10). The tensile strength of PCL strips incubated in sterile wheat inoculated with PCL (A) strain showed its reduction after 144 hours and yet a significant reduction was observed (P< 0.05). Overall, the total tensile strength of PCL strips incubated with PCL (A) strain and PCL (B) strain was lost after 456 hours and 384 hours, respectively.

16.00 14.00

12.00 10.00 Strain A 8.00 Strain B 6.00 Control 4.00

2.00 Tensile Strength (MPa) Strength Tensile 0.00 0 72 144 240 312 384 456

Time (hours)

Figure 2.10 Ability of fungal strains isolated from the surface of PCL in compost at 50°C to degrade PCL strips. PCL strips incubated in wheat and inoculated with fungal strains: PCL A and PCL B at 50°C for 3 weeks. Data represents the means of 3 replicates ± standard error of mean.

2.4.7 Scanning electron microscopy

SEM showed fungal growth on the surface of the polymer discs under all conditions. In order to compare between fungal growth on the surface of the polymers, discs were recovered from compost at 50°C and soil at 25°C. The surface of the discs were scanned and compared to the control discs (Figures 2.11 – 2.14). It is apparent that all scanned discs which were buried under compost and soil at 50°C and 25°C were colonized by a huge mat of fungal growth compared to the control discs which were left unburied at room temperature. Control discs were clear and had no growth at all.

A B C

Figure 2.11 Fungal growth on the surface of PCL discs under controlled conditions visualised using SEM. PCL discs were (A) control, (B) soil condition at 25°C or (C) compost condition at 50°C. Discs buried in compost and soil were colonized by fungal growth, whereas no growth was found on control discs which have been left unburied at room temperature.

A B C

Figure 2.12 Fungal growth on the surface of PLA discs under controlled conditions visualised using SEM. PLA discs were (A) control, (B) soil condition at 25°C or (C) compost condition at 50°C. Discs buried in compost and soil were colonized by fungal growth, whereas no growth was found on control discs which have been left unburied at room temperature.

A B C

Figure 2.13 Fungal growth on the surface of PBS discs under controlled conditions visualised using SEM. PBS discs were (A) control, (B) soil condition at 25°C or (C) compost condition at 50°C. Discs buried in compost and soil were colonized by fungal growth, whereas no growth was found on control discs which have been left unburied at room temperature.

A B C

Figure 2.14 Fungal growth on the surface of PHB discs under controlled conditions visualised using SEM. PHB discs were (A) control disc, (B) soil condition at 25°C or (C) compost condition at 50°C. Discs buried in compost and soil were colonized by fungal growth, whereas no growth was found on control discs which have been left unburied at room temperature.

2.5 Discussion

In order to investigate the rate of degradation of BDPs, polymer weight change was estimated during polymer burial in soil and compost at various temperatures. PCL showed the fastest degradation among the four types of tested polymers at the three temperatures (25°C, 37°C and 50°C) applied to compost and soil under controlled conditions. This was followed by PHB at all temperatures while PLA showed the slowest degradation rate. However, a significant (P<0.05) reduction of PLA weight was observed at 50°C toward the middle of the burial period until complete degradation was recorded in compost at 50°C after 271 days. 72

The rate of biodegradation of BDPs is influenced by a number of parameters including molecular structure, molecular weight, degree of crystallinity and melting temperature of the polymer (Chandra and Rustgi, 1998). Other factors that also might affect the rate of degradation are the characteristics of the incubation environment (soil and compost) in terms of organic and inorganic compositions. Compost is known to have more organic matter than soil but soil can have a higher abundance of inorganic components like minerals and rocks (Manna and Paul, 2000). Moreover differences between polymers, the different types of degrading microbiota and the incubation temperature are all factors that can affect the degradation rate of the polymers (Manna and Paul, 2000; Reddy et al., 2003; Bano et al., 2017).

In this study, PCL was shown to be degraded far more rapidly than PBS, PLA or PHB and thus it can be concluded that PCL is highly susceptible to biodegradation. A number of previous studies have demonstrated rapid degradation of PCL. For example, a study reported that a significant degradation of PCL were observed in compost at 40°C after 35 days (Fukushima et al., 2010b) and Funabashi et al. (2007) have reported that the degree of biodegradability of PCL powder at 47 days reached 90% under controlled compost condition at 58°C. Similar results were reported by Fukushima et al. (2010a) where PCL was fully degraded in compost after 8 weeks. Nishide et al. (1999) also found that PCL showed the fastest degradation rate at 52°C in aerobic soil. Therefore, temperature was clearly highly correlated to the rate of PCL degradation.

In contrast to PCL, little degradation in PLA was observed either in soil or compost at 25°C or 37°C and only a ca. 10% decrease was seen toward the end of the 18 weeks of incubation period at 50°C (Figure 2.3). However, PLA disc weight started to rapidly reduce from week 19 until complete disc degradation was recorded at the end of the incubation period as shown in Figure 2.3A. Fukushima et al. (2009) reported a significant reduction in the molecular weight of PLA when incubated in compost at 40°C for 17 weeks indicating significant depolymerisation although dry weight was not monitored. Moreover, it was shown that a significant weight loss in PLA was not observed until depolymerisation had occurred and complete weight loss was recorded after burial in compost after 6 weeks at 50°C (Karamanlioglu & Robson, 2013), which is well-matched with the results presented here although it took more time for the degradation to occur in this study (19 weeks). A possible explanation for this might be the shape of the polymer (disc shape with average

73 thickness of 3mm) compared to film thickness in Karamanlioglu & Robson (2013). Accordingly, it can suggest that increased sample thickness will greatly reduce the total contact between hydrolytic enzymes and the total surface area of the sample, thereby reducing degradation rate as stated by Yang et al. (2005).

On the other hand, it has been shown from Figure 2.4 that for the PBS polymer, although the results showed no significant decrease in weight in compost at 25°C and 37°C and in soil at 25°C, PBS lost weight significantly in compost at 50°C and soil at 37°C. It has previously been observed that the degradation of PBS, in terms of percentage weight loss and the loss of mechanical properties, was significantly higher in compost soil rather than natural soil because of the increased temperature and humidity conditions in the chamber of the compost (Kim et al., 2006). It has been reported as well that PBS showed the slowest degradation out of the four plastics (in comparison to PHB, PCL and polybutylene succinate and agipate (PBSA)) at 30°C under aerobic conditions (Nishide et al., 1999).

In contrast, PHB polymer showed a significant degradation in soil and compost at all three temperatures; however, the degradation was higher in compost than in soil. Similar findings have shown that the rate of weight loss of PHB can be enhanced by the incubation of the polymer at 40°C (Mergaert et al., 1994). Moreover, a study indicated that a temperature of 46°C facilitated the biodegradation of PHB and PCL by microorganisms that used the polymers as nutrients. It was also reported that there was a greater degradation of PHB at 46°C in soil compost compared to 24°C for the same environment (Lotto et al., 2004). It was found as well that the erosion rate of PHB was more enhanced by incubation the polymer at higher temperatures (Mergaert et al., 1994). These findings suggest that high temperature plays a major role in enhancing the polymer degradation as well as the burial environment where compost acts as a vital environment that can accelerate the biodegradation compared to soil.

Polymer discs that have been buried in natural field conditions showed much more variation in the degradation process. The final weight of each polymer was compared to its original weight before burial. Only PCL showed significant reduction in polymer weight, whereas PHB, PBS and PLA showed no significant reduction. Moreover, some discs showed an increase in final weight like in most discs of PLA and PBS and some discs of PHB. The degrading organisms present in the environment have a very crucial role on the 74 degradation of the BDPs. In addition the studies of biodegradability of polymers in nature and the publication in this field is very rare (Sawada, 1994; Cho et al., 2011; Emadian et al., 2017).

In this study, PCL was the leading polymer for rate of weight loss over the 21 months burial period followed by the PHB polymer. However, most PBS samples and all PLA samples increased in weight. The present study raises the possibility that each polymer requires specific environmental conditions for the biodegradation to occur. Therefore careful consideration regarding the nature and the conditions of the environment where polymers can be disposed or landfilled should be made. It has been stated in a recent review paper that the degradation rate of certain BDP in the natural environment differs from one environment to another (Haider et al., 2019). A previous study found that the biodegradation process of PLA in real soil environment over a period of 11 months was very slow compared to cellulose, which was considered as a positive control under the same environmental conditions. These results were correlated to the low temperature of the real environment and the short duration of the experiment compared to the higher temperature and long duration really needed to degrade the BDPs (Rudnik and Briassoulis, 2011). This is equivalent to the observations here, which showed that the degradation of BDPs can be affected by the soil environment and the temperature. In addition, it has been reported that some types of PLA stay in the soil for a long period of time, and for these forms of PLA to be degraded, they require a high temperature environment such as a composting system (Adhikari et al., 2016). Another study showed that the degradation rate of PHA films at Hoa Lac, Vietnam was more than 98% compared to the same films which lost only 47% of their weight when landfilled in the soil environment of Dam Bai, Vietnam (Emadian et al., 2017). This finding might be related to the relatively low pH (5.48) of Dam Bi soil, which probably reduces the microbial activity (Emadian et al., 2017). Moreover, it has been stated that PLA degradation in soil is slow and takes a long time for the degradation to start (Tokiwa et al., 2009). These results are consistent with those of Ishii et al. (2008) who reported that PBS degradation rate found to be lower than other BDPs such as PCL or PHB in the natural environment and was dependant on the environmental conditions of the area. They further suggested that the degrading strains of PBS are less common than the degrading strains of PCL or PHB. In addition, a review paper has stated that it takes several years for PLA to completely disappear in the environment although PLA is considered as a BDPs (Qi et al., 2017). 75

Moreover it has been stated that the susceptibility of PLA to be degraded naturally in soil and compost is lower than other aliphatic BDPs like PCL (Nampoothiri et al., 2010). Other studies have shown that PLA is less susceptible to microbial attack compared to other polymers and this is because the PLA-degrading microorganisms are not widely distributed in different environments (Pranamuda et al., 1997; Suyama and Tokiwa, 1998; Tansengco and Tokiwa, 1998).

Polymer degrading microorganisms are distributed in the environment and these can be found extensively in many natural habitats like soil, compost, aquatic environment and sludge. Some of these microorganisms has been isolated and identified as biopolymer degraders like Bacillus sp. and Aspergillus sp (Suyama and Tokiwa, 1998; Tokiwa et al., 2009; Adhikari et al., 2016; Emadian et al., 2017). It is well known that BDPs can be used by microbes including bacteria and fungi as a substrate when under starvation and lacking essential nutrition (Bano et al., 2017). Microorganisms will adhere to the polymer surface making a biofilm and become responsible for the degradation of polymers by utilising the hydrocarbons in the backbone of the polymer as a carbon source. Moreover, microorganisms that produce extracellular enzymes for the degradation of polymers are widely distributed in the environment such as soil, water, industrial waste, compost, sludge, marine environments and the human body (Numata et al., 2004; Sukkhum et al., 2009; Li et al., 2012; Bano et al., 2017). Degradation of a number of BDPs including those used in this study have been shown to be mediated by microbial extracellular esterases including lipases and cutinases, causing hydrolysis of the ester linkages and progressive depolymerisation (Nakamura et al., 2001; Numata et al., 2007; Ishii et al., 2008; Shah et al., 2008; Tokiwa et al., 2009).

Fungi isolated from the surface of the polymers at four temperatures in soil and compost were identified by ITS sequencing (Table 2.4). The most prevailing fungus at 50°C on PCL, PHB and PLA was T. lanuginosus. This is a thermophilic fungus that can be found in many habitats like soil and compost (Singh et al., 2003). The strains belonging to this fungus can grow between a maximum temperature of 60°C and a minimum of 20°C with an optimum growth temperature of 50°C (Singh et al., 2003). T. lanuginosus produces heat-stable enzymes that can tolerate high temperatures more than those produced by mesophiles when extracted and tested in cell-free system (Singh et al., 2003). Many studies have emphasised that there is an important need to isolate thermophilic microorganisms that

76 are capable of degrading polymers because of their importance in the composting process, which is considered as one of the most promising technologies in biodegradable polymer recycling (Tseng et al., 2007; Chua et al., 2013). Karamanlioglu et al. (2014) also identified T. lanuginosus as the most frequently recovered isolate from the surface of PLA buried in compost at 50°C. Emadian et al. (2017) confirmed that Thermomyces, Fusarium, Aspergillus and Penicillium are among the main degraders of BDPs. However, there are little reports on the degradation of polymers, and none for PCL, at high temperatures by a fungus (Sanchez et al., 2000). This finding is supported by a very recent report stating that all of the PCL degraders that have been isolated at high temperatures are bacteria (Emadian et al., 2017). Thus, our study is among the first to isolate T. lanuginosus from the surface of PCL at high temperature and consider this fungus as a PCL degrader at 50°C after testing its ability to degrade PCL polymer.

On the other hand, the most dominant fungus at 37°C on all four polymers under soil and compost conditions was A. fumigatus. However at 25°C both A. fumigatus and F. solani were the principal degraders present on PCL and PHB surfaces. These findings were also reported by Chua et al. (2013) where Aspergillus and Fusarium were found to be among the degrading microorganisms that degraded PCL. Another study reported two PCL depolymerases, cutinase and lipase which have been purified from the phyto-pathogen Fusarium (Li et al., 2012). Moreover another study showed that both A. fumigatus and F. solani were isolated from the surface of PLA when incubated at 25°C in soil and compost (Karamanlioglu et al., 2014). Some of the fungal phytopathogens (Fusarium sp.) produce cutinase that degrade a cutin polyester (the structural component of a plant cuticle) (Leja and Lewandowicz, 2010). Tokiwa et al. (2009) has reported that the degrading microorganisms of PBS are abundant in the environment, but their ratio to the total microorganisms is lower than PCL degrading microorganisms (Suyama and Tokiwa, 1998). Moreover, it was estimated that the percentage of PHB degrading microorganisms in the environment was to be 0.5-9.6% of the total colonies and most of these degraders were isolated at mesophilic temperatures. In addition, the same study stated that the microorganisms which were capable of degrading PHB at high temperatures were very few (Suyama and Tokiwa, 1998).

Testing the ability of the identified fungal strain to degrade PCL polymer was performed using the tensile strength technique after adding the PCL strips into wheat grains

77 inoculated with T. lanuginosus strains. It was found that PCL strips in wheat grains inoculated with fungal strains lost tensile strength faster and to a higher extent compared to the strips in uninoculated wheat grains (control). Both sets of PCL strips with fungal strain PCL(A) and PCL(B) showed a significant reduction (P<0.05) in tensile strength measurements. This reduction indicated that T. lanuginosus strains were capable of the degradation of PCL polymer and it is the principle reason for this degradation because no change in the tensile strength were detected in the PCL strips which were incubated in the sterile wheat with no fungal inoculum. However as stated above, there are few reports to identify the degraders of BDPs, and none for PCL at high temperatures by a fungus (Sanchez et al., 2000). The degradability test of the isolates of other polymers would be assessed in future work or in further studies.

Figures 2.11 – 2.14 show before and after images of the degradation surface of the tested polymers in soil, compost and control conditions by scanning electron microscopy. Before the burial (control condition) the surface of the discs exhibited a relative smooth and clear surface. After the burial in soil and compost at different temperatures, there were a number of large holes in the disc surface indicating that the polymer discs were attacked by the microorganisms within the soil and compost environments. This study showed relatively high colonization and surface degradation under compost conditions. The biodegradability of the polymers under compost conditions at 50°C should be more enhanced compared to the biodegradability under soil conditions at 25°C due to the slow rate of the of hydrolysis at low temperatures (Kim et al., 2006).

2.6 Conclusion

With the increasing production and consumption of plastics in daily life, and the consequences of the disposal of these plastics, the need to introduce new plastics that are environmentally friendly is essential. For these environmentally friendly BDPs, there is a need to determine the best disposal method of every BDP. The rate of biodegradation of four BDPs has been investigated in this study under controlled and environmental conditions. Under both conditions PCL polymer was the leading polymer as it degrades more rapidly compared to the other polymers. This study found that temperature was highly correlated to the rate of PCL degradation under controlled conditions. It was clear as well that the type of the burial environment and its conditions such as temperature, pH and microbial activity have a clear and direct effect on the degradation rate of different 78

BDPs. The degrading microorganism have been isolated from the surface of the polymers and they have been identified and observed under SEM. PCL degraders under 50°C conditions have been tested for their biodegradability and T. lanuginosus was found to be the main PCL degrader at this temperature. This study suggested that the isolation of new thermophilic microorganisms is of high importance in order for those microorganisms to be applied for future composting technology.

2.7 References

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Chua, T.-K., Tseng, M. and Yang, M.-K. (2013) ‘Degradation of Poly(ε-caprolactone) by thermophilic Streptomyces thermoviolaceus subsp. thermoviolaceus 76T-2.’ AMB Express, 3(1) p. 8.

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Cosgrove, L., McGeechan, P. L., Robson, G. D. and Handley, P. S. (2007) ‘Fungal communities associated with degradation of polyester polyurethane in soil.’ Applied and Environmental Microbiology, 73(18) pp. 5817–5824.

Crabbe, J. R., Campbell, J. R., Thompson, L., Walz, S. L. and Schultz, W. W. (1994) ‘Biodegradation of a colloidal ester-based polyurethane by soil fungi.’ International Biodeterioration and Biodegradation, 33(2) pp. 103–113.

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Fukushima, K., Abbate, C., Tabuani, D., Gennari, M., Rizzarelli, P. and Camino, G. (2010a) ‘Biodegradation trend of poly(ε-caprolactone) and nanocomposites.’ Materials Science and Engineering C, 30(4) pp. 566–574.

Fukushima, K., Tabuani, D., Abbate, C., Arena, M. and Ferreri, L. (2010b) ‘Effect of sepiolite on the biodegradation of poly(lactic acid) and polycaprolactone.’ Polymer Degradation and Stability, 95(10) pp. 2049–2056.

Funabashi, M., Ninomiya, F. and Kunioka, M. (2007) ‘Biodegradation of polycaprolactone 80 powders proposed as reference test materials for international standard of biodegradation evaluation method.’ Journal of Polymers and the Environment, 15(1) pp. 7–17.

Ishii, N., Inoue, Y., Tagaya, T., Mitomo, H., Nagai, D. and Kasuya, K. I. (2008) ‘Isolation and characterization of poly(butylene succinate)-degrading fungi.’ Polymer Degradation and Stability, 93(5) pp. 883–888.

Karamanlioglu, M., Houlden, A. and Robson, G. D. (2014) ‘Isolation and characterisation of fungal communities associated with degradation and growth on the surface of poly (lactic) acid ( PLA ) in soil and compost.’ International Biodeterioration & Biodegradation, 95 pp. 301–310.

Karamanlioglu, M. and Robson, G. D. (2013) ‘The influence of biotic and abiotic factors on the rate of degradation of poly(lactic) acid (PLA) coupons buried in compost and soil.’ Polymer Degradation and Stability, 98(10) pp. 2063–2071.

Leja, K. and Lewandowicz, G. (2010) ‘Polymer Biodegradation and Biodegradable Polymers – a Review.’ Polish J. of Environ. Stud, 19(November 2009) pp. 255–266.

Lotto, N. T., Calil, M. R., Guedes, C. G. F. and Rosa, D. S. (2004) ‘The effect of temperature on the biodegradation test.’ Materials Science and Engineering: C, 24(5) pp. 659–662.

Manna, A. and Paul, a. K. (2000) ‘Degradation of microbial polyester poly(3- hydroxybutyrate) in environmental samples and in culture.’ Biodegradation, 11(5) pp. 323–329.

Mergaert, J., Anderson, C., Wouters, A. and Swings, J. (1994) ‘Microbial degradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in compost.’ Journal of environmental polymer degradation, 2(3) pp. 177–183.

Nakamura, K., Tomita, T. and Abe, N. (2001) ‘Purification and Characterization of an Extracellular Poly (l-Lactic Acid ) Depolymerase from a soil Isolate , Amycolatopsis sp. Strain K104-1.’ Applied Environmental Microbiology, 67(1) pp. 345–353. 81

Nampoothiri, K. M, Nair, N. R. and John, R. P. (2010) ‘An overview of the recent developments in polylactide (PLA) research.’ Bioresource technology, 101(22) pp. 8493– 501.

Nishide, H., Toyota, K. and Kimura, M. (1999) ‘Effects of soil temperature and anaerobiosis on degradation of biodegradable plastics in soil and their degrading microorganisms.’ Soil Science and Plant Nutrition, 45(4) pp. 963–972.

Numata, K., Hirota, T., Kikkawa, Y., Tsuge, T., Iwata, T., Abe, H. and Doi, Y. (2004) ‘Enzymatic Degradation Processes of Lamellar Crystals in Thin Films for Poly[( R )-3- hydroxybutyric acid] and Its Copolymers Revealed by Real-Time Atomic Force Microscopy.’ Biomacromolecules, 5(6) pp. 2186–2194.

Numata, K., Yamashita, K., Fujita, M., Tsuge, T., Kasuya, K. I., Iwata, T., Doi, Y. and Abe, H. (2007) ‘Adsorption and hydrolysis reactions of poly(hydroxybutyric acid) depolymerases secreted from Ralstonia pickettii T1 and Penicillium funiculosum onto poly[(R)-3- hydroxybutyric acid].’ Biomacromolecules, 8(7) pp. 2276–2281.

Pranamuda, H., Tokiwa, Y. and Tanaka, H. (1997) ‘Polylactide degradation by an Amycolatopsis sp.’ Applied and Environmental Microbiology, 63(4) pp. 1637–1640.

Qi, X., Ren, Y. and Wang, X. (2017) ‘New advances in the biodegradation of Poly(lactic) acid.’ International Biodeterioration & Biodegradation, 117, February, pp. 215–223.

Reddy, C. S. K., Ghai, R., Rashmi and Kalia, V. C. (2003) ‘Polyhydroxyalkanoates: An overview.’ Bioresource Technology, 87(2) pp. 137–146.

Rudnik, E. and Briassoulis, D. (2011) ‘Degradation behaviour of poly(lactic acid) films and fibres in soil under Mediterranean field conditions and laboratory simulations testing.’ Industrial Crops and Products, 33(3) pp. 648–658.

Sanchez, J. G., Tsuchii, A. and Tokiwa, Y. (2000) ‘Degradation of polycaprolactone at 50 °C by a thermotolerant Aspergillus sp.’ Biotechnology Letters, 22(10) pp. 849–853.

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Chapter 3

Characterisation of polycaprolactone as a promising biodegradable polymer

3.1 Abstract

Biodegradable polymers (BDPs) have received extensive attention because of their faster degradation in the environment. Polycaprolactone (PCL) is a polymer which attracted interest as a potential substitute of conventional polymers because of its properties as a synthetic polymer that can be biodegradable. PCL was tested for its degradation characteristics in two different polymer forms: polymer strips and polymer powder. The results showed that PCL strips has a significant reduction in tensile strength with time when measured after incubation in compost at four different temperatures. Moreover, for distributed polymer samples such as powders, the reduction in residual polymer following compost incubation was also significant. It was confirmed that PCL degradation rate increases with time and as temperatures increases in an environment rich in degrading microorganisms such as compost.

3.2 Introduction

BDPs are considered to be ‘green’ polymers because of their limited negative impact on the environment, and therefore they are considered as an alternative to conventional plastics. Many BDPs have been developed to date such as PCL, polyhydroxybutyrate (PHB), polylactic acid (PLA), and polybutylene succinate (PBS) (Ohtaki et al., 1998; Shimao, 2001; Funabashi et al., 2007; Tokiwa et al., 2009; Ahmed et al., 2018). However, the safety and applications of these polymers are based on how they will be disposed off and how the society will handle them (Rujnić-Sokele and Pilipović, 2017).

Among these degradable polymers, PCL is a thermoplastic synthetic polymer which is synthesized by ring-opening polymerisation of ε-caprolactone. This polymer has attracted special attention because of its properties including flexibility and biodegradability (Khatiwala et al., 2008; Ahmed et al., 2018). In addition, PCL is well known to have compatibility with many other polymers because of its properties which makes it a good substrate and an attractive polymer for many applications (Fukushima et al., 2010; Borghesi et al., 2016; Vivi et al., 2019). Furthermore, PCL is a partially-crystalline polyester which has a low melting point (60°C) and glass transition temperature of -60 °C (Shimao, 2001; Funabashi et al., 2007; Tokiwa et al., 2009; Borghesi et al., 2016). It also has chemical resistance to water, oil, solvent and chlorine. This polymer has low viscosity and it is easy to process (Funabashi et al., 2007; Siracusa et al., 2008). These physical properties makes PCL very attractive because it can be used for many applications in medicine and in agricultural domains (Vivi et al., 2019).

The biodegradation of PCL has been studied intensively and received much attention in order to assess its disposal in the environment and for its applications. Many studies have reported that PCL is degradable in many natural biotic environments such as soil, compost, seawater and active sludge (Li et al., 2012; Nawaz et al., 2015; Borghesi et al., 2016). The hydrolysable ester linkage in the chemical structure of this linear aliphatic polyester makes it susceptible to microbial degradation (Albertsson et al., 1998). This polymer can be degraded by lipase and esterase enzymes and its degrading microbes can be found distributed widely in the environment (Albertsson et al., 1998; Ahmed et al., 2018).Moreover, when pure cultures of different strains of Fusarium were used to degraded PCL, the results showed that Fusarium strains degraded the polymer with

86 cutinase and then the degraded PCL was used as carbon source for the microorganism (Murphy et al., 1996; Rutkowska et al., 2002).

Following on from the results presented in Chapter 2, this chapter is focused on the degradation rate of PCL polymer in different forms (strips and powder) with time to determine the characterisation of PCL as a promising biodegradable polymer.

3.3 Materials and Methods

3.3.1 PCL strip compost incubation and tensile strength measurement over time

1. PCL strips

PCL strips were prepared as sheets form by melting 12.5 g of PCL pellets in 100 ml of dichloromethane (DCM) solvent. The melted PCL was poured into a leveled glass cast and left to set as a sheet with an even thickness (0.4 – 0.5 mm). The sheet was then cut into strips (6 cm x 0.5 cm) for the sampling experiment.

2. Compost preparation

Commercial compost were obtained from the Compost shop (UK) and prepared using a 7 mm sieve prior to use. The percentage moisture content was calculated by drying ca. 1 g of compost in triplicate at 55°C to a constant weight. The percentage moisture content was calculated and was found to be 33.19%.

3. Compost extraction

The method for compost extraction was followed as described previously (Karamanlioglu and Robson, 2013) with some modification. 250 g of compost was mixed with 250 ml water. The mixture was left at room temperature for 1 hour with occasional shaking then the supernatant was filtered with filter paper twice to remove any large particles.

4. PCL strips burial in compost

Rectangular plastic boxes were filled with 400 ml compost and 40 pre-sterilized PCL strips were added to each box and buried in the compost. The strips were pre-sterilized by washing in 70% ethanol. The boxes then were incubated at four different temperatures: 25°C, 37°C, 45°C, or 50°C for 10 weeks.

There were two types of control: 87

- Four PCL strips were kept dry in a petri dish at each temperature condition to be measured at the end of the burial period or when the strips in the box disappeared. - Four PCL strips were kept in compost extract at each temperature condition to be measured at the end of the burial period or when the strips in the box disappeared.

Moreover, four PCL stripes were also measured at the start of the experiment as a 0 week reading.

The buried strips were measured once a week for the 25°C and 37°C treatments, and twice a week for the 45°C and 50°C treatments to have multiple measurements over time for each temperature treatment. At each time point, four PCL strips were recovered, any compost sticking to the strips was removed and the tensile strength of the strips was measured using a T-series Tensile Test Machine (Tinius Olsen, LTD. Surrey, UK) supported with QMAT Professional software.

3.3.2 PCL powder compost incubation and remaining residual measurement over time

1. Compost preparation

Compost was prepared as described above (Section 3.1); it was sieved to remove any large particles, and the moisture content was calculated and found to be 33.19%.

2. PCL /compost mixture preparation:

In rectangular plastic boxes, 400 ml of compost were mixed with 40 ml of PCL powder which has 50,000 molecular weight and particle size of < 600 µm (to give 10% PCL in the compost mixture). Holes were made on the lid of each box and sealed with parafilm to allow gas exchange and aerobic respiration. The boxes were incubated at five different temperatures 25°C, 37°C, 45°C, 50°C and 55°C for eight weeks and control boxes were incubated without PCL powder. Samples (3 g) were recovered every week from three different random locations within each box and were pooled together in universal tubes.

3. Measuring the remaining PCL residual from the compost/PCL mixture

From each compost /PCL mixture tube incubated at each of the five temperatures, 1 g was taken off and mixed with 5 ml DCM solvent in a glass tube and left overnight. The

88 next day a capillary glass pipette was used to remove the solvent from the glass tube (avoiding any soil particles) and the solvent was transferred to a glass petri dish and left until the PCL solidified to produce plastic sheet. The weight of the produced sheet was measured. This was repeated at each weekly time point for eight weeks for each of the temperature treatments.

3.4 Results

3.4.1 PCL strip compost incubation and tensile strength measurements over time

Tensile strength measurements were used to test the degradation rate of PCL polymer under compost conditions at four temperatures from 25°C to 50°C. Figures 3.1 – 3.4 show the degradation rate of PCL with time at different temperatures and report the significant decrease in tensile strength for the polymer with time at each temperature treatment.

2 0

)

P M

( 1 5

h t

g * n

e 1 0 r

t *

l * i 5

s * n

e * T 0

0 1 2 3 4 5 6 7 8 9 0 t l 1 c o a r r t t n x o e t C s o p m o C

T im e (w e e k s )

Figure 3.1 Tensile strength measurements of PCL strips over time at 25°C. Each bar represents the mean of four replicates of PCL strips that were buried in compost and measured over ten weeks. PCL strips incubated with a compost extract and control strips incubated in the absence of compost were measured after ten weeks. The error bars represent the standard error of mean. An asterisk (*) represents significant difference in comparison to Week 0 value (P <0.05).

2 0

)

P M

( 1 5

g n

e 1 0 *

t S

e * l

i 5 *

s n

e * T 0

0 1 2 3 4 5 6 7 8 9 0 t l 1 c o a r r t t n x o e t C s o p m o C

T im e (w e e k s )

Figure 3.2 Tensile strength measurements of PCL strips over time at 37°C. Each bar represents the mean of four replicates of PCL strips that were buried in compost and measured over ten weeks. PCL strips incubated with a compost extract and control strips incubated in the absence of compost were measured after ten weeks. The error bars represent the standard error of mean. An asterisk (*) represents significant difference in comparison to Week 0 value (P <0.05).

There was a significant decrease in the tensile strength measurements of PCL polymer with time under 25°C and 37°C treatments as shown in Figures 3.1 and 3.2, especially at the end of the burial period (Week 10) when the tensile strength reached 0.51 MPa after 25°C incubation and 1.31 MPa after 37°C incubation compared to the starting Week 0 value, which was 14.18 MPa. Control strip incubation in compost extract or dry (without any compost addition) in a petri dish showed no significant change in the tensile strength compared to the Week 0 value.

Figure 3.3 presents the tensile strength measurements of PCL strips incubated in compost at 45°C. Again a significant reduction was shown with time but for this treatment the decrease in tensile strength was significant after just two weeks of incubation. Again there was no significant change in the control strips. At 45°C the measurements could only be taken for eight weeks because of the fast degradation of the strips as no intact strips could be recovered after eight weeks.

) 2 0

M (

1 5

n e

r 1 0

t *

e l i 5 s * n * e * * *

T * 0 t l 0 1 2 3 4 5 6 7 8 c o a r tr t x n e o t C s o p m o C

T im e (w e e k s )

Figure 3.3 Tensile strength measurements of PCL strips over time at 45°C. Each bar represents the mean of four replicates of PCL strips that were buried in compost and measured over eight weeks. PCL strips incubated with a compost extract and control strips incubated in the absence of compost were measured after eight weeks toward the end of the burial period. The error bars represent the standard error of mean. An asterisk (*) represents significant difference in comparison to Week 0 value (P <0.05).

2 0

)

P M

( 1 5

g n

e 1 0

r t

S *

e l

i 5 s

n * e

T * * 0

0 1 2 3 4 5 t l c o a r r t t n x o e t C s o p m o C

T im e (w e e k s )

Figure 3.4 Tensile strength measurements of PCL strips over time at 50°C. Each bar represents the mean of four replicates of PCL strips that were buried in compost and measured over five weeks. PCL strips incubated with a compost extract and control strips incubated in the absence of compost were measured after five weeks. The error bars represent the standard error of mean. An asterisk (*) represents significant difference in comparison to Week 0 value (P <0.05). 91

The degradation rate of PCL strips at 50°C declined drastically from Week 2 (Figure 3.4). By Week 5 the tensile strength value had reached 0.44 MPa. PCL stripes could only be recovered for five weeks due to the fast degradation of PCL polymer under this higher temperature, and no intact strips could be recovered after five weeks. Again there was no significant change in the control strips.

3.4.2 PCL powder compost incubation and remaining residual measurements over time.

In order to measure how much PCL residual remained with respect to incubation time and temperature in compost, a 1 g triplicate sample from a 10% PCL compost mix was tested at each time point over eight weeks following five different temperature treatments ranging from 25°C to 55°C. The amount of residual PCL was compared with the amount present at the start of the experiment (Week 0).

Figure 3.5 shows the PCL residual remaining at 25°C over time. There was a significant reduction in PCL sheet weight compared to the 100% PCL sheet weight (Time 0) from Week 1 onwards. However this reduction was moderate and far smaller than the reduction in weight of PCL sheets under each of the higher temperatures up to 50°C (Figures 3.6, 3.7 and 3.8). The 45°C and 50°C treatments in particular gave significant reductions in PCL sheet weight over time, suggesting that at these temperatures PCL would be totally degraded with time and that high temperatures play a key role in the degradation of PCL polymer.

It was therefore surprising that the degradation of PCL at 55°C was much slower although still showed a significant reduction after Week 1, but with very minor reduction over time, and more equivalent to the degradation pattern at 25°C (Figure 3.9).

g n

i 1 5 0

L 1 0 0

l * * a * *

u * * d i 5 0 *

s *

a 0 e

M 0 1 2 3 4 5 6 7 8

T im e (w e e k s )

Figure 3.5 Mean percentage of PCL residual remaining with time at 25°C. Each bar represents the mean of three replicates of PCL remaining in compost collected from three random locations within a mixture box containing 10% PCL in compost. The error bars represent the standard error of mean (not visible due to very small values). An asterisk (*) represents significant difference in comparison to Week 0 value (P <0.05).

g n

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L 1 0 0

l * a * u *

d * i 5 0

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e r

% *

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T im e (w e e k s )

Figure 3.6 Mean percentage of PCL residual remaining with time at 37°C. Each bar represents the mean of three replicates of PCL remaining in compost collected from three random locations within a mixture box containing 10% PCL in compost. The error bars represent the standard error of mean (not visible due to very small values). An asterisk (*) represents significant difference in comparison to Week 0 value (P <0.05).

g n

i 1 5 0

L 1 0 0

l *

u d i 5 0 s *

e * r

* %

* *

n * *

a 0 e

M 0 1 2 3 4 5 6 7 8

T im e (w e e k s )

Figure 3.7 Mean percentage of PCL residual remaining with time at 45°C. Each bar represents the mean of three replicates of PCL remaining in compost collected from three random locations within a mixture box containing 10% PCL in compost. The error bars represent the standard error of mean (not visible due to very small values). An asterisk (*) represents significant difference in comparison to Week 0 value (P <0.05).

g n

i 1 5 0

L 1 0 0

l *

u d

i 5 0 * s

e * r

* * *

% * *

a 0 e

M 0 1 2 3 4 5 6 7 8

T im e (w e e k s )

Figure 3.8 Mean percentage of PCL residual remaining with time at 50°C. Each bar represents the mean of three replicates of PCL remaining in compost collected from three random locations within a mixture box containing 10% PCL in compost. The error bars represent the standard error of mean (not visible due to very small values). An asterisk (*) represents significant difference in comparison to Week 0 value (P <0.05).

g n

i 1 5 0

L 1 0 0

l * * * * a *

u * * d

i 5 0 *

a 0 e

M 0 1 2 3 4 5 6 7 8

T im e (w e e k s )

Figure 3.9 Mean percentage of PCL residual remaining with time at 55°C. Each bar represents the mean of three replicates of PCL remaining in compost collected from three random locations within a mixture box containing 10% PCL in compost. The error bars represent the standard error of mean (not visible due to very small values). An asterisk (*) represents significant difference in comparison to Week 0 value (P <0.05).

3.5 Discussion

The weight loss of test specimens such as polymer films is widely used to determine the degradation of polymers. Moreover, for distributed polymer samples such as powders , the reduction in residual polymer can be determined by polymer extraction from the soil or compost using a solvent extraction technique (Shah et al., 2008). Both methods have been used in this chapter to further quantify the degradation of PCL polymer after the results of the previous chapter indicated that this polymer gave the most promising results.

It was shown that PCL polymer tensile strength declined with time. At 25°C, 37°C and 45C°, the polymer showed significant degradation with time and it reached 0.51 MPa at 25°C after ten weeks, 1.31 MPa at 37°C after ten weeks and 2.53 MPa at 45°C after eight weeks. However PCL polymer tensile strength reached its lowest value (0.44 MPa) at 50°C in Week 5. This might be because the polymer is experiencing more microbial degradation and becomes weak over time which makes its tensile strength decrease with time, as indicated in some previous studies (Shimao, 2001; Fukushima et al., 2010). PCL films treated as a control in compost extract or without compost or water addition

95 showed no significant change and this indicates that the polymer was been degraded due to microbial action but not due to hydrolytic disintegration. Moreover, as the temperature increased during the compost incubation, the ability for measuring the tensile strength over longer time periods was less as the polymer strips began to disintegrate more rapidly under higher compost temperatures. Nevertheless, it was not temperature alone that was increasing PCL disintegration since the control strips with compost at each temperature showed no change in strength. A study showed that the degradation of PCL in a biotic environment was faster compared to an abiotic environment, and the degradation rates were much higher in complex environment like compost due to the cooperation between highe temperature and the presence of rich microbial community (Albertsson et al., 1998). It was reported by Fukushima et al. (2010) that there was a significant surface degradation of PCL films after three weeks buried in compost at 50°C. It has also been reported that PCL was completely degraded by a strain of Aspergillus sp. after 6 days incubation in soil at 50°C (Tokiwa et al., 2009). It is worth mention as well that the activity of the enzymes is dependant on the temperature, and at relatively lower tempertaure like 30°C the the ectivity of the enzyme will be lowered by 60% (Haider et al., 2019).

The second method that was used in this study to evaluate the degradation rate of the PCL polymer was the measurement in residual polymer. The results showed significant reduction in residual polymer over time in compost under all five temperatures that were used. However, there was substantial variation in the amount of PCL reduction. There was greater reduction at higher temperatures except at 55°C. We might relate this low reduction in polymer degradation at 55°C to the potential reduced abundance of microbial communities within the compost that can tolerate the higher temperature of 55°C. For example, few species of fungi have the ability to survive at temperatures between 45°C and 55°C (Maheshwari et al., 2000). Again this would indicate the importance of microbial activity rather than increased temperature itself for the degradation of the PCL polymer. It has been stated that the combination of high temperature together with high abundance microorganisms can lead to higher degradation rate compared to degradation due to high temperature alone with low microbial comminuty presence (Albertsson et al., 1998). In addition, it has been stated that PCL polymer is considered as a relatively stable polymer against abiotic hydrolysis,

96 however it can be easily attacked and degraded by microorganisms that are distributed in the environment (Shimao, 2001; Fukushima et al., 2010).

3.6 Conclusion

PCL was the leading polymer for further characterisation identified in the prevoius chapter and so was exposed to two different methods to evaluate its degradation rate. Two forms of the polymer were tested: polymer strips and polymer powder. Under both methods PCL was tested for its degradation over time under different temperatures. it can be concluded that PCL degradation rate in compost increased with time and as we move to higher temperatures in the presence of microbiota. All results showed significant reduction in tensile strength and in the remaining in residual polymer. However, there was a slowing in degradation activity at a temperature of 55°C.

3.7 References

Ahmed, T., Shahid, M., Azeem, F., Rasul, I., Shah, A. A., Noman, M., Hameed, A., Manzoor, N., Manzoor, I. and Muhammad, S. (2018) ‘Biodegradation of plastics: current scenario and future prospects for environmental safety.’ Environmental Science and Pollution Research. Environmental Science and Pollution Research, 25(8) pp. 7287–7298.

Albertsson, A.-C., Renstad, R., Erlandsson, B., Eldsater, C. and Karlsson, S. (1998) ‘Effect of processing additives on (bio)degradability of film-blown poly(Ɛ-caprolactone).’ Journal of Applied Polymer Science, 70(1) pp. 61–74.

Borghesi, D. C., Molina, M. F., Guerra, M. A. and Campos, M. G. N. (2016) ‘Biodegradation Study of a Novel Poly-Caprolactone-Coffee Husk Composite Film.’ Materials Research, 19(4) pp. 752–758.

Fukushima, K., Abbate, C., Tabuani, D., Gennari, M., Rizzarelli, P. and Camino, G. (2010) ‘Biodegradation trend of poly(ε-caprolactone) and nanocomposites.’ Materials Science and Engineering C, 30(4) pp. 566–574.

Funabashi, M., Ninomiya, F. and Kunioka, M. (2007) ‘Biodegradation of polycaprolactone powders proposed as reference test materials for international standard of biodegradation evaluation method.’ Journal of Polymers and the Environment, 15(1) pp. 7–17.

Khatiwala, V. K., Shekhar, N., Aggarwal, S. and Mandal, U. K. (2008) ‘Biodegradation of Poly(ε-caprolactone) (PCL) Film by Alcaligenes faecalis.’ Journal of Polymers and the Environment, 16(1) pp. 61–67.

Maheshwari, R., Bharadwaj, G. and Bhat, M. K. (2000) ‘Thermophilic fungi: their physiology and enzymes.’ Microbiology and molecular biology reviews : MMBR, 64(3) pp. 461–88.

Murphy, C. A., Cameron, J. A., Huang, S. J. and Vinopal, R. T. (1996) ‘Fusarium polycaprolactone depolymerase is cutinase.’ Applied and environmental microbiology, 62(2) pp. 456–60.

Nawaz, A., Hasan, F. and Shah, A. A. (2015) ‘Degradation of poly(ε-caprolactone) (PCL) by a newly isolated Brevundimonas sp. strain MRL-AN1 from soil.’ FEMS Microbiology Letters, 362(1) pp.1-7.

Ohtaki, A., Sato, N. and Nakasaki, K. (1998) ‘Biodegradation of poly-ε-caprolactone under

98 controlled composting conditions.’ Polymer Degradation and Stability, 61(3) pp. 499–505.

Rujnić-Sokele, M. and Pilipović, A. (2017) ‘Challenges and opportunities of biodegradable plastics: A mini review.’ Waste Management and Research, 35(2) pp. 132–140.

Shah, A. A., Hasan, F., Hameed, A. and Ahmed, S. (2008) ‘Biological degradation of plastics: A comprehensive review.’ Biotechnology Advances, 26(3) pp. 246–265.

Shimao, M. (2001) ‘Biodegradation of plastics.’ Current Opinion in Biotechnology, 12(3) pp. 242–247.

Siracusa, V., Rocculi, P., Romani, S. and Rosa, M. D. (2008) ‘Biodegradable polymers for food packaging: a review.’ Trends in Food Science & Technology, 19(12) pp. 634–643.

Tokiwa, Y., Calabia, B. P., Ugwu, C. U. and Aiba, S. (2009) ‘Biodegradability of plastics.’ International Journal of Molecular Sciences, 10(9) pp. 3722–3742.

Vivi, V. K., Martins-Franchetti, S. M. and Attili-Angelis, D. (2019) ‘Biodegradation of PCL and PVC: Chaetomium globosum (ATCC 16021) activity.’ Folia Microbiologica, 64(1) pp. 1– 7.

Chapter 4

The impact of polycaprolactone degradation on microbial communities in compost at different temperatures using next generation sequencing

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4.1 Abstract

Fungal communities in nature are diverse, some are free living, others symbiotic, and some are plant or animal pathogens. Unicellular yeasts are also part of the fungal communities present in nature and in general fungi are considered to comprise the largest amount of biomass in comparison to other organisms. Compost microbial communities are well known to be involved in the degradation of polymers. However, differences in the type of polymers and the abiotic conditions present in the surrounding environment could have an influence on the fungal community involved in decomposition process.

The sequencing of DNA extracted from environmental samples, offers a unique opportunity to study whole microbial communities in situ. The use of molecular approaches for microbial community analysis provides a more comprehensive investigation of the polymer degradation process facilitated by various fungal taxa.

In this study, the effect of the degradation of polycaprolactone (PCL) polymer on the compost microbial communities has been studied. The compost microbial communities were analysed using the next generation sequencing and bioinformatics methods.

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4.2 Introduction

Microbial communities and their diversity have been investigated using many different traditional and molecular methods (Cosgrove et al., 2007; Lim et al., 2010; Klindworth et al., 2013; Kim et al., 2014; Nam et al., 2015; Aguinaga et al., 2018). It is very important to study any change or shift in microbial communities because these variations in the microbial community structure can influence ecosystem processes. Quantification of microbial communities can represent a powerful tool for understanding community dynamics in an ecological context (Agrawal et al., 2015).

There are different methods for studying microbial diversity and one of these methods is next generation sequencing technologies (NGS). These NGS technologies can allow researchers to use relatively short DNA sequences, typically of ribosomal RNA gene sequences, to identify vast numbers of organisms from environmental samples (De Beeck et al., 2014). Moreover, analysis of the diversity and abundance of whole microbial communities, and the identification of the potential function of those communities, can all be quantified by these NGS technologies at far greater depth than traditional methods (Lemos et al., 2017).To the best of our knowledge there are few studies on the impact of polymer degradation in soil and compost on microbial community structure. For example a study has found that after five months incubation of the polyester polyurethane (PU) in soil, the fungal communities associated with the PU were less diverse than the communities in the soil (Cosgrove et al., 2007). In addition, another study on the impact of polylactic acid (PLA) hydrolysis on fungal communities in compost has found that after two months incubation of PLA in compost at 50°C, a high shift in the fungal community structure was found in the presence compared to the absence of PLA (Karamanlioglu et al., 2017).

Therefore in this study, the impact of polycaprolactone (PCL) degradation on compost microbial communities at different temperatures has been investigated. PCL is known to be an artificially synthesized polymer that is synthesized by ring-opening polymerization of cyclic e-caprolactone. PCL is also known to be a biodegradable and compostable polymer that can be degraded under natural environmental conditions including in soil and compost by the action of microorganisms (Lotto et al., 2004; Fukushima et al., 2010; Li et al., 2012).

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PCL along with other biodegradable polymers (BDPs) has been used as a substitution for conventional polymers in many applications such as medical, agricultural and packaging applications (Funabashi et al., 2007; Li et al., 2012). However, it is very important to understand the possible changes that might occur within microbial communities in soil and compost if these biodegradable polymers are released and then degraded within the environment.

The aim of this study is to use NGS technique to quantify fungal community structures under different conditions and to investigate if PCL degradation in compost can affect compost microbial communities structure. In this study 10% PCL was mixed with compost samples and incubated under different conditions to test any changes in the microbial community structures and compare it with compost samples with no PCL added at ambient temperature.

4.3 Materials and Methods

4.3.1 Compost preparation

Compost was sieved to remove any large particles, and the moisture content was calculated and found to be 33.19%.

4.3.2 PCL /compost mixture preparation

In rectangular plastic boxes, 400 ml of compost was mixed with 40 ml of PCL powder (this makes 10% PCL in compost mixture). Holes were made on the lid of each box and sealed with parafilm to allow gas exchange and aerobic respiration.The boxes were incubated at five different temperatures 25°C, 37°C, 45°C, 50°C and 55°C for eight weeks and control boxes were incubated at each temperature without PCL powder. Samples were recovered every week from three different random sites of each box, around 1 g of PCL/compost mixture was taken from each site and was pooled in universal tubes. The tubes were stored in -80C until further analysis needed.

4.3.3 DNA extraction from compost

0.25g of compost/PCL mixture and of control compost was extracted for its DNA using the Powersoil® DNA Isolation kit (MO-BIO Laboratories, CA, USA) according to the manufacturer’s instructions. There were two time points for each temperature and for

103 each time point there was a compost/PCL sample and a control sample from which DNA was extracted.

4.3.4 Next generation sequencing and data analysis

This work was carried out at the Integrated Microbiome Resource (IMR) centre for Comparative Genomics and Evolutionary Bioinformatics (CGEB), Dalhousie University, Canada. The extracted DNA samples were sent for gene amplification and sequencing of the ITS2 region of fungal 5.8S and 26S rRNA gene sequence, which was amplified by the use of the primers:

ITS86F = 5’-GTGAATCATCGAATCTTTGAA-3’

ITS4R = 5’-TCCTCCGCTTATTGATATGC-3’

The amplicon samples were run on Illumina MiSeq platform using 300+300 bp paired-end V3 chemistry. The barcode sequences were removed from the ends of the reads, and the sequence reads were provided as raw data. The analysis of fungal reads were performed using the PIPITS pipeline (Gweon et al., 2015), which needs a number of third-party applications for different steps of the analysis (Wang et al., 2007; McDonald et al., 2012; Bengtsson-Palme et al., 2013; Zhang et al., 2014; Rognes et al., 2016). The reads were stitched to each other by examining the overlapping regions of the sequences. The resulting assembled reads were then quality filtered according to the following filtering criteria: reads that had at least 80 % of their nucleotides with a quality score of 30 were kept. Also the sequences which contained unknown nucleotides were kept. The sequences which belonged to the ITS2 region were kept for the next steps. Additionally, short (< 100bp) and unique sequences were removed prior to finding operational taxonomic unites (OTUs). The sequences then were clustered at a threshold of 97% sequence identity. The resulting representative sequences for each cluster were subjected to chimera detection and removal using the UNITE uchime reference dataset (https://unite.ut.ee/repository.php). The input sequences were then mapped onto the chimera-free representative sequences at the defined threshold. The representatives were taxonomically assigned with RDP Classifier in conjunction with Warcup training set against the UNITE fungal ITS reference dataset with the confidence threshold of 0.85.

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The data were analysed using Megan6 software, excel and R software. Megan6 software was used to open and read BIOM format files (The Biological Observation Matrix) of the raw data and extract these data to be used for producing graphs and tables. Excel was used to produce tables and bar graphs. R software was used to produce rarefaction curves and Principal component Analysis graphs (PCA). Rarefaction data and graphs were produced by using ‘vegan package’. PCA was performed using ‘ggfortify package’ and the graphs were drawn via ‘ggplot2 package’.

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4.4 Results

In order to quantify fungal diversity, numbers of OTUs were determined from the sequence read data. Time points at Week 5 and 8 were analysed. These two time points were chosen according to our findings in Chapter 3 where higher degradation of PCL occurred from Week 5 onward and as usually little degradation had been observed before this week. Moreover, this time point represents the middle of the incubation period. On the other hand, week 8 time point have been chosen for the analysis because this time point represents the end of incubation period. Table 4.1 shows the number of fungal OTUs and the total number of reads calculated for samples incubated at 5 different temperatures for 5 or 8 weeks.

Table 4.1 Number of fungal OTUs and the total number of reads calculated for samples incubated at five different temperatures for five or eight weeks. PCL indicates the compost with PCL and the number of each sample name indicates the time point in weeks.

Time point No of Total No of Sample Temp (C°) (week) OTUs reads PCL5 5 25 89 21215 PCL8 8 25 153 67790 Control5 5 25 246 73924 Control8 8 25 262 69028 PCL5 5 37 138 70249 PCL8 8 37 173 110016 Control5 5 37 137 45068 Control8 8 37 151 69750 PCL5 5 45 86 62225 PCL8 8 45 89 31899 Control5 5 45 65 56829 Control8 8 45 125 37150 PCL5 5 50 26 60580 PCL8 8 50 28 72574 Control5 5 50 44 19430 Control8 8 50 19 437 PCL5 5 55 39 6020 PCL8 8 55 41 79412 Control5 5 55 42 936 Control8 8 55 30 498 Ambient Time 0 Control 0 temperature 260 57373

106

The highest number of fungal sequences per sample was 110,016 and this was found in sample PB8 which is compost with PCL at 37°C after eight weeks (Table 4.1). These number of sequences accounted for 173 OTUs. On the other hand, the lowest number of sequences per sample was 437 and it was found in CD8 sample which is control compost (with no PCL) at 50°C at Week 8. This number of sequences accounts only for 19 OTUs. The highest number of OTUs per sample was 262 in sample CA8, which is control compost at 25°C sampled at Week 8. This number of OTUs is almost the same as in the initial compost sample (260 OTUs) and this indicates the general lack of change in the microbial community on the basis of OTU number at 25°C. This can be quantified by the following double logarithmic graph (Fig. 4.1) which shows a linear regression analysis for log (OTU number) against log (temperature).

8 C o n tr o l

P C L s

U 6

o 4

)

n L

( 2

0 3 .0 3 .5 4 .0 4 .5 (L n )T e m p e ra tu re

Figure 4.1 Linear regression analysis of the correlation between temperature and number of OTUs. Both control data and compost with PCL data showed a negative relationship. The black line shows the relationship for both treatments combined. The numbers used in this graph are represented as natural log.

Linear regression analysis demonstrated that there is a significant (P<0.05) correlation between temperature and OTU numbers both for control data and for data of compost with PCL. It was clear that as the temperature increased, OTU numbers decreased which indicates a strong negative relationship.

Table 4.2 displays two diversity indices: Shannon-Weaver index and Simpson index of diversity. 107

Table 4.2 Diversity indices calculated for all fungal samples incubated at five different temperatures for five or eight weeks. PCL indicates the compost with PCL and the number of each sample name indicates the time point in weeks.

Time point Shannon-Weaver Simpson index of Sample Temperature(C°) (weeks) index diversity PCL5 5 25 2.27 0.83 PCL8 8 25 2.48 0.87 Control5 5 25 2.21 0.80 Control8 8 25 2.49 0.84 PCL5 5 37 2.50 0.89 PCL8 8 37 2.54 0.89 Control5 5 37 2.16 0.82 Control8 8 37 1.94 0.79 PCL5 5 45 1.77 0.77 PCL8 8 45 2.13 0.81 Control5 5 45 1.12 0.59 Control8 8 45 1.72 0.73 PCL5 5 50 0.16 0.05 PCL8 8 50 0.19 0.07 Control5 5 50 0.38 0.12 Control8 8 50 2.28 0.86 PCL5 5 55 0.40 0.12 PCL8 8 55 0.09 0.02 Control5 5 55 2.58 0.85 Control8 8 55 2.69 0.90 Time 0 0 Control 2.20 0.81 Control

The Simpson index of diversity can measure the species richness in a given community. The value of this index ranges between 0 and 1, where the greater the value, the greater the diversity. Hence it can be seen from the data in Table 4.2 that all samples are very diverse and rich except for the control sample at Week 5 incubated at 50°C, and all PCL samples at 50°C and 55°C. Likewise, the Shannon-Weaver index is another measure of species diversity in a community and it accounts for both abundance and evenness of the species present. Values of the Shannon diversity index for real natural communities typically fall between 1.5 and 3.5. Table 4.2 shows that all of the samples that show low Simpson index values also have low Shannon-Weaver index values. These results indicate that microbial diversity can be affected in compost in the presence of PCL at high temperatures.

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A B

C D

Figure 4.2 Rarefaction curves of observed fungal OTUs richness for compost at different temperatures at 97% sequence similarity. A: compost at 25°C, B: compost at 37°C, C: compost at 45°C, D: compost at 50°C and E: compost at 55°C.

Rarefaction analysis of the OTUs at 97% similarity showed that sequence read saturation was reached by all of the compost samples at 25°C, 37°C and 45°C, which revealed a high fungal richness in the compost samples, while samples that did not show high species richness from the 50°C and compost at 55°C samples did not saturate, indicating that additional sequence reads may have been needed to determine if all OTUs were captured (Figure 4.2).

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Figure 4.3 Principal Component Analysis (PCA) of fungal communities isolated from initial compost (Time 0 control) and compared with compost containing 10% PCL and compost control at Week 5 and 8 at five different temperatures. A: compost at 25°C, B: compost at 37°C, C: compost at 45°C, D: compost at 50°C and E: compost at 55°C.

The potential variance in the fungal community data sets was examined by Principal Component Analysis (PCA). At every temperature there were differences between the control compost sample at Week 0 and nearly all of the samples collected at Week 5 and 8 (Figure 4.3). At 25°C, two principal components (PC1 and PC2) accounted for 88.5% of

110 the total variability in the data set (Figure 4.3A). Differences between the initial compost sample and compost with PCL at Week 5 and 8 can be seen clearly along PC1 axis, which explained 53.2% of the variation, while control compost at week 5 and 8 differed from the initial compost on the basis of PC2. For PCL incubated in compost at 37°C, PC1 accounted for 77.4% of the variation and explained the difference between the control compost samples without PCL and the compost samples with 10% PCL at week 5 and 8. In contrast, differences between Week 5 and 8 were seen along the PC2 axis, which accounted for 15% of the variation (Figure 4.3B).

At 45°C, the first two principal components (PC1 and PC2) accounted for 89.9% of the total variability in the fungal community (Figure 4.3C). PC1 showed mainly the difference between compost samples with and without PCL at Week 5, and to a lesser degree at Week 8, while PC2 mainly explained the difference between compost samples over time. Figure 4.3D showed the PCA of fungal communities isolated from compost samples incubated at 50°C. Both PC1 and PC2 accounted for 97.6% of the total variability within the dataset. PC1 accounted for 89.2% of the total variation and it highlighted the difference between the compost samples with and without 10% PCL regardless of time point. In contrast, PC2 mostly explains the difference between the initial compost sample and the samples either with or without PCL at the later time points.

At 55°C where both PC1 and PC2 accounted for 99.9% of the total variability in the fungal community, PC1 showed the difference between the 10% PCL at Week 8 and all other samples, while PC2 again mostly explains the difference between the initial compost sample and the samples either with or without PCL at the later time points (Figure 4.3E). Figure 4.4 to 4.8 represent the relative abundance of fungal taxa at order level from compost samples with 10% PCL and without PCL (control compost) at two time points at each of the five different temperature treatments and compared with the initial compost at the control (ambient) temperature. This initial control compost sample showed approximately 60% abundance of Sordariales and 35% abundance of Eurotiales along with very small amounts of other taxa.

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100

80 Mortierellales

70 Saccharomycetales

60 Orbiliales

50 Sordariales

40 Microascales

30 Hypocreales Relative abundance (%) abundance Relative

20 Glomerellales

10 Eurotiales

0 Time 0 PCL5 Control5 PCL8 Control8 Control Samples

Figure 4.4 Relative abundance of fungal sequences (order level) from compost samples with (10%) PCL and compost control at two time points (Weeks 5 and 8) at 25°C compared with the initial compost (time 0 control) at ambient temperature.

As shown in Figure 4.4, the taxonomic assignment of fungal OTUs revealed that in compost samples that have been incubated at 25°C, the order with highest number of sequences was Eurotiales, followed by Hypocreales, and Sordariales, respectively. Eurotiales was 53% abundance in control8 (higher than the initial compost) and Hypocreales was highly abundant in sample PCL8 with a value of 61%. While abundance of Sordariales was highest in the initial compost sample the abundance of this taxa was markedly reduced in all other samples, particularly in the presence of PCL. Further changes in the microbial structure can be seen under each compost sample where for example Orbiliales increased in PCL5 to 36% where it was only 0.7% abundant in the initial compost. The abundance of Orbiliales was also higher in PCL8 sample in contrast to the samples without PCL. Moreover, Hypocreales was abundant in all of the Week 5 and 8 samples but almost absent in the initial compost. Other taxa were present at very low abundance across the samples.

112

100

80 Eremomycetaceae

Mortierellales 70 Saccharomycetales 60 Orbiliales 50 Sordariales

40 Microascales

30 Hypocreales Relative abundanc (%) abundanc Relative Eurotiales 20

0 Time 0 PCL5 Control5 PCL8 Control8 Control

Samples

Figure 4.5 Relative abundance of fungal sequences (order level) from compost samples with (10%) PCL and compost control at two time points (Weeks 5 and 8) at 37°C compared with the initial compost (time 0 control) at ambient temperature.

Figure 4.5 shows that, the fungal orders with highest abundance for the samples incubated at 37°C are the same orders that were identified at 25°C; they are Eurotiales, followed by Hypocreales, and Sordariales, respectively. Eurotiales was highly abundant in all samples, especially in the control sample at Week 5 where it accounts for 51% of the total abundance. Hypocreales was present highly in both time points of PCL samples, while Sordariales was very low in both PCL samples. However, Eremomycetaceae appeared with relatively high abundance in PCL5 and PCL8 with 21% and 18% abundance, respectively. Other taxa were present at very low abundance across the samples, including Orbiliales, which was present at much lower abundance in comparison to the PCL samples at 25°C.

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100

80 Mortierellales Agaricales 70

Saccharomycetales 60 Orbiliales

50 Sordariales Microascales 40 Hypocreales 30 Thelebolales

20 Eurotiales Relative abundance (%) abundance Relative Dothideales 10

0 Time 0 PCL5 Control5 PCL8 Control8 Control Samples

Figure 4.6 Relative abundance of fungal sequences (order level) from compost samples with (10%) PCL and compost control at two time points (Weeks 5 and 8) at 45°C compared with the initial compost (time 0 control) at ambient temperature.

The most abundant orders present at 45°C are again Sordariales, Eurotiales and Hypocreales (Figure 4.6). In contrast to the lower temperature treatments (25°C and 37°C) there were high percentages of Sordariales present among all samples at 45°C with approximately 50% abundance, except in the control5 sample, which displayed only 21% abundance of this order. Eurotiales order was found with high abundance in the PCL8 sample, equivalent to the initial compost sample. On the other hand, Hypocreales had a more variable distribution between the samples in contrast to the previous temperature, this order present in the control5 sample with 60% abundance but with relatively low abundance in the other samples. Agaricales, which had not been detected at the lower temperatures, showed a high percentage abundance in PCL5 (23%) and a lower abundance (4%) in PCL8. Other taxa were present at very low abundance.

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100 Mortierellales 90 Sporidiobolales 80 Trichosporonales Tremellales 70 Atheliales 60 Agaricales 50 Saccharomycetales Orbiliales 40 Sordariales 30

Coniochaetales Relative abundance (%) abundance Relative 20 Microascales

10 Hypocreales Eurotiales 0 Pleosporales Time 0 PCL5 Control5 PCL8 Control8 Control

Samples

Figure 4.7 Relative abundance of fungal sequences (order level) from compost samples with (10%) PCL and compost control at two time points (Weeks 5 and 8) at 50°C compared with the initial compost (time 0 control) at ambient temperature.

At 50°C there were very distinct profiles between the samples (Figure 4.7). Eurotiales was highly abundant, with values of 99% and 98 % in the PCL5 and PCL8 samples, respectively, but almost absent in the control5 sample, and with relatively low abundance in the other control samples. In contrast, Hypocreales dominated the control5 sample (95% abundance) and was present at 47% in the control8 sample. Finally, Sordariales was largely absent in all of the samples at this temperature in comparison to the initial compost. Some other taxa were present in these samples at low abundance including Tremellales and Trichosporonales in control8 with 17% and 4% abundance, respectively. These two orders did not appear in the other compost samples at this temperature.

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100.00

90.00 Mucorales 80.00

Mortierellales

70.00 Malasseziales

60.00 Trichosporonales Saccharomycetales 50.00 Orbiliales 40.00 Sordariales

30.00 Microascales Relative abndance (%) abndance Relative Hypocreales 20.00 Eurotiales 10.00 Pleosporales 0.00 Time 0 PCL5 Control5 PCL8 Control8 Control Samples

Figure 4.8 Relative abundance of fungal sequences (order level) from compost samples with (10%) PCL and compost control at two time points (Weeks 5 and 8) at 55°C compared with the initial compost (time 0 control) at ambient temperature.

As shown in Figure 4.8, the samples at 55°C also had very distinct profiles between the samples with and without PCL. While the PCL samples at 50°C were dominated by Eurotiales, at 55°C Hypocreales had 99% and 96% abundance in PCL8 and PCL5 samples, respectively. In contrast, Sordariales was the most abundant order control5 and control8 samples, as it also was in the initial compost sample. Eurotiales had considerably low abundance in the samples at this temperature compared to the lower temperatures. Other taxa were present in some of the samples at low abundance including Mortierellales, found in control5 and control8 with 15% and 7%, respectively but was virtually absent in the initial compost.

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100 A 90

70 60 50 Sordariales Hypocreales 40 Eurotiales 30

Relative abundance (%) abundance Relative 20 10 0 Control 25°C 37°C 45°C 50°C 55°C Temperature (C°)

B 100 90

80 70 60 50 Sordariales 40 Hypocreales 30 Eurotiales 20

Relative abundance (%) abundance Relative 10 0 Control 25°C 37°C 45°C 50°C 55°C Temperature (C°)

Figure 4.9 Relative abundance of fungal sequences for the highest dominant taxa orders present in compost samples at all five temperatures. A: represents the three highest dominant taxa orders in compost samples with PCL at Week 5 (time point five PCL5). B: represent the three highest dominant taxa orders in compost samples with PCL at Week 8 (time point eight PCL8). Both A and B are compared against control (initial compost sample) at ambient temperature.

Figure 4.9 shows the three most dominant taxa orders present in the compost samples containing PCL at all temperatures compared to the initial compost sample without PCL at ambient temperature. It is clear that the Sordariales order showed very low abundance at 25°C and 37°C at both time points in response to PCL, had intriguingly higher abundance at 45°C but almost absent at 50°C and 55°C for both time points. On the other hand, Hypocreales order, which was almost absent in the initial compost showed a relatively small increase in abundance at 25°C, 37°C, 45°C and 50°C then dominated the samples at 55°C. Finally, Eurotiales showed a substantial increase in abundance at 50°C but then was virtually absent at 55°C. 117

80000

70000

60000

50000

40000 Thermomyces 30000

Talaromyces No.of reads reads No.of 20000

10000

PE5 PE8

CE5 CE8

PC5 PC8

PB5 PB8

CC5 CC8

CB5 CB8

PA5 PA8

CA8 CA5

PD5 PD8

CD5 CD8 Control Samples

Figure 4.10 Number of reads of fungal sequences of genus levels below Eurotiales order for all compost samples used in this study. P: indicates PCL, C: indicates Control, the letters indicates temperatures: A(25°C), B (37°C), C (45°C), D (50°C) and E (55°C). The numbers indicates the time points of each sample in weeks (Week 5 and Week 8).

It is obvious from Figure 4.10 that the most consistently dominant genus below the Eurotiales order was Thermomyces sp. which can be found in all compost samples at all temperatures where it accounts for 139,061 total numbers of sequence reads, and is particularly abundant in samples up to 45°C. Although Talaromyces sp. was only dominant in PD5 and PD8 samples (with PCL at 50°C), the total number of sequence reads of this genus was 132,603 which was almost equivalent to the overall Thermomyces sp. abundance.

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90000

80000

70000 Ophiocordyceps 60000 Hirsutella 50000 Nectria 40000

30000 Emericellopsis No. of reads reads ofNo. 20000 Hypocreaceae

10000 Lecanicillium

PE5 PE8

CE5 CE8

PC5 PC8

PB5 PB8

CC5 CC8

CB5 CB8

PA5 PA8

CA5 CA8

PD5 PD8

CD5 CD8 Control Samples

Figure 4.11 Number of reads of fungal sequences of genus levels below Hypocreales order for all compost samples used in this study. P: indicates PCL, C: indicates Control, the letters indicates temperatures: A(25°C), B (37°C), C (45°C), D (50°C) and E (55°C). The numbers indicates the time points of each sample in weeks (Week 5 and Week 8).

Results from Figure 4.11 show that the most dominant genus below Hypocreales order was Ophiocordyceps sp. which was present in all of the compost samples at all temperatures, although most abundant in sample PE8, and it accounts for 245,247 sequence reads. This was followed by Nectria sp. which mostly dominant in samples at 25°C, particularly PA8, and was detected through 6,184 numbers of sequence reads.

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30000

Neurospora 25000 Podospora

20000 Thielavia

Myceliophthora 15000 Humicola

10000 Chaetomium No. of reads reads ofNo. Cephalotheca 5000

PE5 PE8

CE5 CE8

PC5 PC8

PB5 PB8

CC5 CC8

CB5 CB8

PA5 PA8

CA5 CA8

PD5 PD8

CD5 CD8 Control Samples

Figure 4.12 Number of reads of fungal sequences of genus levels below Sordariales order for all compost samples used in this study. P: indicates PCL, C: indicates Control, the letters indicates temperatures: A(25°C), B (37°C), C (45°C), D (50°C) and E (55°C). The numbers indicates the time points of each sample in weeks (Week 5 and Week 8).

The highest total number of sequence reads below the Sordariales order was 35,405 and these were for Chaetomium sp. (Figure 4.12). This was followed by Thielavia sp. which accounts for 3,056 sequence reads. Chaetomium sp. was mainly found in the compost samples at 45°C in the presence of PCL.

4.5 Discussion

After the removal of short and poor quality sequences, 1,012,403 sequences were obtained in the Illumina MiSeq run for the 21 samples analysed in this study. The final OTU table contained 2,243 OTUs. The highest number of sequences per sample was 110,016 and this was found in sample PB8 which is compost with PCL at 37°C at Week 8. These number of sequences accounts for 173 OTUs. On the other hand, the lowest number of sequences per sample was 437 and it was found in CD8 sample which is control compost (with no PCL) at 50°C at Week 8. This number of sequences accounts only for 19 OTUs. The highest number of OTUs per sample is 262 and it was found in sample CA8 which is control compost at 25°C at Week 8, this number of OTUs was

120 equivalent to the OTU number in the initial compost sample which was 260 and this indicates the minor change in the microbial community at 25°C. However, this does not mean that the makeup of the taxa is the same. Therefore, it can be said that, the number of OTUs decrease with increasing temperature as has been shown by the linear regression analysis. It is worth mentioning that this study has got some limitations that could not be avoided and this led to the low number of reads which makes data at 50°C and 55°C to be considered as unreliable data and hence conclusion should be drawn only based on 45°C, 37°C and 25°C.

DNA concentration extracted from compost samples at 50°C and 55°C were very low and considered as not eligible to undergo through MiSeq run for sequencing, however the sequencing was performed anyway. The DNA extractions were repeated many times and yet got the same results. Due to time constrained the experiment and hence the extraction cannot be repeated. For all these reasons, data at 50°C and 55°C should be discussed with caution. On the other hand, this low DNA concentration might be related to the reduced abundance of microorganisms within the compost that can tolerate these higher temperatures. For example, it has been stataed that only few fungal species have the capability to survive at temperatures between 45°C and 55°C (Maheshwari et al., 2000).

A diversity index is a mathematical measure of species diversity in a given community. Diversity indices provide more information about community composition than simply species richness. They also take the relative abundances of different species into account (Gheno-Heredia et al., 2016). In this study diversity indices revealed that most of the samples showed high species diversity and richness. Five samples extracted at higher temperatures e.g. 50°C and 55°C stood out as having low diversity. Besides that, four out of five of these samples included PCL. It might be concluded that the presence of PCL in compost at higher temperatures might shift or lower the microbial diversity especially at 55°C. It is well known that at 55°C microbial diversity is typically much lower compared to other temperatures because low number of species can tolerate this high temperature as mentioned before (Maheshwari et al., 2000). It has been stated in the literature that only a few species of fungi can survive at temperatures between 45°C and 55°C (Maheshwari et al., 2000; Langarica-Fuentes et al., 2014). Hence, with the presence of PCL, the microbial diversity becomes lower.

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Rarefaction analysis of the OTUs assigned at 97% similarity can also be used to confirm the diversity results where the rarefaction results revealed that most of the samples have approached or are close to the plateau zone which indicates high diversity and richness. The high diversity can be seen clearly in compost at 25°C, 37°C, and 45°C. However the microbial communities are less diverse in compost at 50°C and 55°C and this might be because fewer microbes are present at this high temperature.

Statistical comparison of the fungal community structure by PCA indicated that at each temperature, the distribution of the fungal communities was based on the presence of PCL (Figure 4.3). Under all temperatures it was clear that control compost samples were separated from compost samples with 10% PCL and almost all control (no PCL) samples were closely clustered to the initial compost samples.

Taxonomic assignment of fungal OTUs revealed significant difference in the fungal community’s organization at the different temperature conditions that have been tested. At the order level, the taxa with the highest number of sequences at all temperatures were Eurotiales, Hypocreales and Sordariales. These three orders were dominant at all five temperatures but with different abundance. All of these fungal sequences belong to the Ascomycota group (Klaubauf et al., 2010). Ascomycota and Basidiomycota members have been found to be as the main soil fungal decomposers as demonstrated by several studies (Ma et al., 2013). The main aim was to compare the compost samples with PCL with the initial compost sample and investigate any difference and shift in the fungal communities structure with the presence of PCL at two time points, Week 5 and Week 8.

It was clear that while Hypocreales order was within the initial compost with very low abundance (0.8%), the relative abundance of this order increased dramatically at 55°C to over 95% at Weeks 5 and 8. The order Hypocreales (Ascomycetes), are ecologically and economically important because it is a saprobic, entomopathogenic, and mycoparasitic fungus. It is also considered be the most important biocontrol agents for agriculture (Chaverri and Vílchez, 2006).

On the other hand, Sordariales appeared in the initial compost as the most dominant order with 60% abundance but its abundance decreased at 25°C and 37°C, increased again at 45°C before it was almost absent at 50°C (0.02%) and 55°C (0.42%). Both Hypocreales and Sordariales belong to the same Sordariomycetes class (Zhang et al.,

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2006). Sordariomycetes is considered as one of the largest monophyletic clades in the Ascomycota because it has more than 600 genera and 3000 known species (Kirk et al., 2001). It can be seen that both Hypocreales and Sordariales are found in the same conditions but they are dominating at different temperatures. This can be due to many characteristics and preferences of the fungal species found within each order group. A study showed that members of the Sordariomycetes class are ubiquitous and can be found in most ecosystems from normal conditions to harsh ones. These members are pathogens and endophytes of plants, arthropods and mammals, and are also considered as nutrient decomposers (Zhang et al., 2006). Another study showed that Sordariomycetes encompasses 16 orders in three sub classes that are Hypocreomycetidae, Sordariomycetidae and Xylariomycetidae and this division is based on rRNA gene phylogeny (Zhang et al., 2006).

Eurotiales on the other hand, was present in the initial compost with 36% abundance, and its abundance varies between 20% and 30% in the 25°C, 37°C and 45°C treatments. However at 50°C the relative abundance of this order reached 99% and almost disappeared at 55°C. Eurotiales is one of the most common groups of fungi in soil which belongs to class Eurotiomycetes, phylum Ascomycota. Most of these groups of fungi are saprotrophic and they have the ability to produce diverse sets of toxic secondary metabolites such as aflatoxins, ochratoxins and patulins, which make these fungi important agents of food spoilage (Geiser et al., 2006). Some species of this order are capable of growing at extreme conditions such as at low water activities i.e. they are xerotolerant and/or osmotolerant. Some are also capable to survive at low temperatures (psychrotolerant) and at high temperatures (thermotolerant) (Geiser et al., 2006; Houbraken et al., 2014).

All of these orders belong to Ascomycota group. A study has found that it is very usual to find the majority of fungal sequences belonged to the Ascomycota group in soil habitats lacking ectomycorrhizal host plants (Schadt et al., 2003). There is more than one variable that can influence the presence of certain fungal communities in one condition and disappear from another condition. Light, moisture, nutrient substrates and temperatures are some factors that can affect the shift in any fungal community (Chaverri and Vílchez, 2006). However, there is little information in the literature about the Ascomycota orders Eurotiales, Hypocreales and Sordariales and their habitats and where these groups are

123 typically living, while there are a number of studies that have examined the taxonomy classification of these groups and their phylogenetic analysis (Morgenstern et al., 2012; Wang et al., 2016). Most of the changes in the fungal community structure from this study can be seen in the compost samples with PCL.

The data of the three dominant orders (Eurotiales, Hypocreales and Sordariales) was analysed at the genus level. Unfortunately taxa could not be identified at species level because the database does not show species level for the taxonomy. Under the Eurotiales order it was found that Thermomyces was the most dominant genus and it was found in all compost samples under all temperatures. In contrast Talaromyces was dominant at compost samples with PCL at Weeks 5 and 8 at 50°C (PD5 and PD8). On the other hand, Ophiocordyceps represented the most dominant genus under the Hypocreales order which was present in all the compost samples at all temperatures. Finally under the Sordariales order, the genus Chaetomium had the highest number of reads and was mainly found in compost samples at 45°C in the presence of PCL.

The literature describes four species belonging to Thermomyces: these are T. lanuginosus, T. ibadanensis, T. stellatus, and T. verrucosus and the phylogenetic studies showed that these species belong to different families and this will be well-suited to our study. T. lanuginosus and T. ibadanensis are true thermophiles, T. stellatus is thermotolerant and T. verrucosus are mesophilic (Morgenstern et al., 2012; Houbraken et al., 2014). Therefore, this can explain the presence of Thermomyces genus in the compost samples under all temperatures. Moreover, there is one species belongs to Talaromyces which is considered to be thermotolerant and this is Talaromyces leycettanus (Morgenstern et al., 2012; Houbraken et al., 2014). This may explain the presence of Talaromyces in compost samples at 50°C in this study.

Ophiocordyceps is a heterogeneous genus in the order Hypocreales (Sordariomycetes, Ascomycota) that includes invertebrate-pathogenic taxa (Sung et al., 2007; Ban et al., 2015; Luangsa-ard et al., 2018). This genus is rich with species that can reach 223 accepted species names (Spatafora et al., 2015). Ophiocordyceps are distributed globally mostly in the tropics and subtropics where the highest number of species has been reported. Ophiocordyceps species found specifically in exposed environment such as in the leaf litter, on the underside the leaves, and buried under the ground (Ban et al., 2015; Luangsa-ard et al., 2018). This can be related to this study in a way that the compost that 124 has been used here had high leaf litter and growth of some plants. This can confirm the high abundance of Ophiocordyceps genus in our data results.

Additionally, Chaetomium species are known to belong to a large genus which is a saprobic ascomycetes. This genus can be found on dung, seeds, plant debris, straw and soil. This genus (Chaetomium) includes more than 400 species and most of them can grow between 25 and 35°C (Barron et al., 2003; Wang et al., 2016). Nevertheless, Asgari and Zare (2011) has stated that one Chaetomium species called Chaetomium jodhpurense has been proved to be a thermotolerant fungus. The optimum temperature for the growth of this fungus is between 30-40°C, however it can still grow at a maximum temperature of 45°C and minimum of 15°C (Asgari and zare, 2011). These findings related to the present study findings where Chaetomium was mostly dominating in compost samples at 45°C. Previous studies evaluating the genus Chaetomium have found that members of this genus are able to colonize different types of substrates and also capable to degrade cellulose and produce various bioactive metabolites (Wang et al., 2016).

Very little was found in the literature on the effect of BDPs on microbial communities in soil and compost to the best of our knowledge except for a few. A study on the microbial communities associated with the biodegradation of the polyester polyurethane (PU) has stated that the fungal communities on the PU surface were different from the surrounding compost community (Zafar et al., 2013). Another study showed that the fungal diversity and community structure in compost was highly affected by the presence of the polymer PLA at 50°C after two months but this shift returned toward the initial community structure after four months, which suggested that at 50°C PLA causes a temporal shift in the microbial community (Karamanlioglu et al., 2017).

Finally, this study suggested that temperature can be considered as a major factor that can drive the structure of the compost microbial community. However, when this factor was combined with the presence of PCL as a secondary factor, additional effects can be seen. This finding was also reported by Zafar et al. (2014) where they found that the structure of the fungal community that was recovered from Polyester Polyurethane buried in a compost pile was dependant on the incubation temperature. Moreover, it can be suggested as well that fungal community structure might not be necessarily directly involved in the degradation of the polymer. A study has found that the products that are produced by the breakdown from the primary colonizers can act as a carbon source for 125 non-degrader microbes and this can explain the presence of the non-degraders on the surface of the polymer (Cosgrove et al., 2007).

4.6 Conclusion

This study has extended our knowledge of fungi community structure with the potential for PCL and temperature to shift or effect the community structure under different environmental conditions. However, the ability of PCL to make a big shift and impact on the compost microbial community has not yet been exploited to its full potential, and should be investigated further.

The results showed that both temperature and the presence of PCL have an effect on the structure of fungal microbial communities. However, it can be seen as well from the findings of this study that fungal community structures have variations in different conditions, and hence the main factor that has driven this shift in the structure needs to be determined by further investigations.

Moreover, fungal community structure might not be necessarily directly involved in the degradation of the polymer but it might be indicating more about a broader compost community structure when this polymer is present rather than the fungi that are directly driving the degradation. There might be only small amount of fungi that is doing the degradation and as yet a lot of these taxa have not been classified.

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Chapter 5

The effect of polycaprolactone degradation in compost on seed germination

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5.1 Abstract

Extracts from compost prepared with different concentrations of degraded polycaprolactone (PCL) under a range of temperature conditions was tested for its effect on the germination of seeds from four species of edible plants: cress, lettuce, mustard, and rocket. The results showed that there was no effect of PCL on cress seed germination in low temperature treatments (below 50°C) and low PCL concentrations (below 5% PCL). However, results showed that in compost prepared at 55°C with 10% and 5% PCL, there was a significant negative effect, where no seed germinated in 10% PCL treatments and only 53.3% of seed germinated in 5% PCL treatments. A similar inhibition of seed germination was seen for lettuce, mustard, and rocket. It can be concluded that under high temperature with high PCL concentrations, seed germination will be inhibited.

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5.2 Introduction

In recent decades, humans are generating plastic wastes, which are accumulating in the environment and potentially causing toxicity. It has been stated that 57 million tonnes of plastic wastes are generated every year, with a large proportion having poor degradation characteristics and will therefore be a cause of long term pollution (Nair et al., 2016).

Biodegradable polymers (BDPs) are therefore considered as an environmental solution to plastic pollution. These polymers are designed to degrade following disposal due to the action of naturally abundant microorganisms. The degradation of these BDPs is proposed to be environmentally acceptable since it should not lead to the generation of toxic or harmful chemicals (Krasowska et al., 2016). BDPs can be broken down in the environment by the action of various abiotic factors such as temperature, humidity, pH, sunlight, alongside the action of microorganisms and their enzymes (Song et al., 2009; Krasowska et al., 2016; Emadian et al., 2017). However, the degradation rates differ from one polymer to another depending on the environment (Haider et al., 2019). Many of these biodegradable processes can be enhanced through composting, which is considered as one of the most efficient waste treatment processes for BDPs. Composting provides fast degradation of organic matter in the presence of microbial populations in an aerobic environment under controlled conditions such as temperature. This natural process will end up producing compost as a major product along with water and CO2 (Song et al.,

2009; Zafar et al., 2013; Kumar and Maiti, 2016; Qi et al., 2017). Natural or amended compost environments are thus a means to help in the elimination of plastic waste by acting as a repository for the degradation of BDPs.

An example of BDPs is PCL. This polymer can undergo microbial degradation in different natural environments and under different conditions. PCL is fully biodegradable when composted since the low melting point (58-60°C) of PCL makes this material suited for composting as a means of disposal (Rudnik, 2010). Due in part to these characteristics, this BDP is increasingly being used as an alternative to conventional plastics for different applications such as in medical and agricultural applications (Krasowska et al., 2016). However, despite the increase in the amount of PCL produced and used, to the best of our knowledge no study has yet examined the effect of PCL degradation in compost on seed germination. The degradability of the polymer is not the only parameter that needs be taken into account when replacing conventional plastics with BDPs but it is also 133 important to consider any adverse effects that the BDP might cause on the biota. The aim of this study was therefore to investigate whether PCL degradation in compost had any effect on seed germination. Different concentrations of PCL were mixed with compost and incubated at different temperatures for eight weeks in order to emulate the natural environment and the different stages of the composting process. Extractions of these compost/PCL mixtures were then used for seed germination tests.

5.3 Materials and Methods

5.3.1 Compost analysis

Commercial compost was obtained (The Compost Shop, UK) and prepared using a 7 mm sieve prior to use. The percentage moisture content was calculated by drying ca. 1 g of compost in triplicate at 55°C to constant weight. The percentage moisture content was calculated and was found to be 33.19 %. The water holding capacity was also measured and found to be 80%, and the compost pH was 7.1.

5.3.2 Compost preparation with 10% PCL

For a first experiment, 400 ml of compost was mixed with 40 ml of PCL powder which has 50,000 molecular weight and particle size of < 600 µm (Polysience,Inc.) in rectangular 1 L plastic boxes (Sealfresh, Stewart, UK) with dimensions 16.5 cm x 11.5 cm x 5 cm to give a 10% PCL in compost mixture. Holes were made in the lid of each box and sealed with parafilm to allow gas exchange and aerobic respiration. Control boxes were also prepared without any added PCL powder. The 10% PCL and control compost boxes were incubated at five different temperatures, 25°C, 37°C, 45°C, 50°C and 55°C, for eight weeks.

5.3.3 Compost preparation with different PCL concentrations

For a second experiment, the above method (Section 5.3.2) was repeated with different PCL concentrations in the compost: 10%, 5%, 2%, 1%, and 0.5%, as well as control boxes without PCL powder. For this experiment, only three temperatures were used: 45°C, 50°C and 55°C. All boxes were incubated for eight weeks.

5.3.4 Compost extract preparation

Following eight weeks incubation at different temperatures, the control and PCL- containing compost was processed to generate a compose extract. The method for

134 compost extraction was followed as described previously (Karamanlioglu and Robson, 2013) with some modification. 250 g of compost was mixed with 250 ml sterilised water then the mixture was left at room temperature for 1 hour with occasional shaking. The supernatant was then filtered with filter papers twice to remove any unwanted particles.

5.3.5 Cress seed germination

Cress seeds were chosen for initial germination experiments due to their fast germination and rapid growth. Seeds were germinated on an oval shape cotton pads that can be obtained from any cosmetic shop in UK. These pads were placed in a petri dish and saturated with approximately 12 ml of compost extract. Triplicate cotton pads were treated with each type of compost extract. 50 seeds were added onto each cotton pad and all of the pads were maintained at room temperature in a well-lit lab room, and were sprayed with sterile water daily to avoid drying out. The germination was determined by sprouting and put forth shoots from the seeds. The number of germinated seeds was counted daily over a 3 day period after which nearly all the seeds had been germinated and the length of the seedling was measured by centimetre using a ruler. The same set up were used with two other controls; compost without PCL, which had been incubated under all temperature conditions and extracted to test its ability to germinate cress seeds. For a non-compost extract control, the cotton pads were treated with water and then used for seed germination.

5.3.6 Germination of different seed types in compost with 10 % PCL at 55°C.

An experiment was performed to compare germination of three different types of seeds (wild rocket, mustard and lettuce) in 10% PCL-compost extract incubated at 55°C, using the same germination method as described above (Section 5.3.5) and compare it with cress seed germination.

5.4 Results

Seed germination (determined as a percentage of all seeds sown) and seedling length (determined after growth at Day 3) were quantified following addition of extracts from composts that had been prepared under different temperature conditions and additions of different PCL concentrations, in order to investigate if there was any effect of PCL degradation on the germination and growth of seeds. 135

5.4.1 Cress seed germination in compost extract with 10% PCL concentration

During an initial experiment, cress seeds were germinated in extract from compost containing 10% PCL in comparison to an extract from compost with no added PCL. The compost had been incubated for eight weeks, to allow the PCL to be degraded, and at five different temperatures ranging from 25°C to 55°C.

As can be seen in Figure 5.1A, cress seeds were able to germinate with nearly 100% efficiency in all control compost extracts that were prepared under all temperature conditions. There was also a very high percentage of germination with 10% PCL compost extracts prepared at 25°C to 50°C, with no significant difference between control compost treatments (Figure 5.1A). For these treatments, the average percentage seed germination varied between 96% to 98%. However, when exposed to extract from PCL-containing compost prepared at 55°C, cress seeds showed no germination.

No seedling length could obviously be determined for the 55°C PCL treatment since no seeds germinated (Figure 5.1B). The seedling length of cress seeds (which represent cress seedling growth) showed no significant difference between all treatments, which indicates that neither the presence of PCL nor the temperature had affected the growth of the cress seedlings. The average seedling length ranged between 2.6 cm to 3.2 cm at all temperatures.

A further experiment was designed to assess the effect of different concentrations of PCL in compost on seed germination.

136

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Figure 5.1 Cress seeds tested in compost extract from compost with 10% PCL at five different temperatures in comparison to control compost. (A) Seed germination determined as a percentage of total cress seeds after three days. (B) The average seedling length measured after three days. Each bar in (A) and (B) represents the mean data of three replicates and the error bars represent the standard error of the mean. An asterisk indicates significant difference (P <0.05) between control and PCL treatment. (N.D) means not determined.

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5.4.2 Cress seed germination in compost extract with different PCL concentrations

The purpose of this experiment was to test the ability of cress seeds to germinate in extracts from compost prepared under three different relatively high temperatures (45°C, 50°C and 55°C) and five different PCL concentrations. As the initial results (Section 5.4.1) showed that cress seeds were unable to germinate in compost extract in the presence of 10% PCL at 55°C, it was decided to assess seed germination in different PCL concentrations to investigate whether any concentration of PCL was able to supress seed germination in compost extract at 55°C.

Figure 5.2A shows cress seed germination in extracts derived from different PCL concentrations at three different temperatures. It was clear that once again there was no cress seed germination at 55°C at all in extract from compost with 10% PCL. Moreover, there was a significantly low percentage of cress seed germination in compost extract with 5% PCL at 55°C. For the PCL concentrations of 2%, 1% and 0.5% at 55°C, seed germination percentage was not significantly different from the control. On the other hand, the germination under 45°C and 50°C under all PCL concentrations were high and equivalent to control.

Cress seedling length showed variation even among the same temperature (Figure 5.2B). Seedling length averages were 3.5 cm, 2.7 cm and 2.4 cm for compost extracts at 45°C, 50°C and 55°C, respectively. It can be clearly seen that compost extract at 45°C can be considered as an optimum temperature for the seed germination and growth among this set of temperatures because both the seed germination and seedling length was high under all PCL concentrations. At 50°C compost extract, the growth has increased from 10% PCL to 2% PCL, then it went down from 1% to 0.5% PCL in compost extract. At 55°C compost extract, the seedling length reached the highest length at 2% PCL. However, seedling length decreased in control compost. 5% PCL in compost extract at 55°C showed the lowest length (average of 1 cm) perhaps because the germination under this condition was also very low (53.3%) compared to the other treatments.

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Figure 5.2 Cress seeds tested in compost extract with different PCL concentrations under three relatively high temperatures in comparison to control compost. (A) Seed germination determined as a percentage of cress seeds after three days. (B) The average seedling length measured after three days. Each bar in (A) and (B) represents the mean data of three replicates and the error bars represent the standard error of mean. An asterisk indicates significant difference (P <0.05) between control and PCL treatment. (N.D) means not determined.

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5.4.3 Germination of different seed types in response to 10% PCL compost extract

Since both PCL concentration and temperature had no effect on cress seed germination in compost extract at temperatures below 55°C, for a final experiment the germination of different types of seeds were tested in compost extract prepared with 10% PCL at 55°C only in order to investigate whether this conditions effect only cress seed germination or whether it can affect other types of seed. These seeds are of wild rocket, mustard and lettuce.

100 90

80 70 60 50 PCL (10%) 40 Control 30

Seed germination (%) germination Seed 20 10 N.D N.D N.D 0 Wild Rocket Mustard Lettuce

Seed types

Figure 5.3 Germination of wild rocket, mustard and lettuce seeds in compost extract prepared at 55°C in 10% PCL in comparison to control compost. Each bar represents the mean data of three replicates and the error bars represent the standard error of mean. An asterisk indicates significant difference (P <0.05) between control and PCL treatment. (N.D) means not determined.

Figure 5.3 illustrated that there was no germination in the 10% PCL compost extract prepared at 55°C for all three types of seeds, whereas in control compost the germination was between 93% to 95%. These results can conclude that the presence of PCL in compost with concentration of at least 10% at 55°C will inhibit seed germination. The temperature has no effect on seed germination because we can still find seed germinating in extract from 55°C control compost.

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5.5 Discussion

Seed germination was tested in compost extract with degraded PCL at different concentrations and incubated at different temperatures. The results showed that PCL had no effect on the germination and growth of cress seeds under all temperatures except at 55°C when PCL concentration is 5% and above. These results indicated that PCL can be an environmentally friendly polymer with no effect on seed germination and seedling growth if found in compost under relatively low temperature conditions.

Usually compost can reach high temperature during industrial composting process as stated by Doi et al. (1992) and the temperature can reach up to 65°C. Composting can produce a high amount of heat due to microbial respiration, however the composting process starts with an initial phase where the temperature rises from ambient temperature to around 45°C (Ishii et al., 2000). Temperature continues to rise until it reaches the thermophilic phase where the temperature is around 70°C. Finally the temperature will go back to the ambient temperature (Ishii et al., 2000).

Although ecotoxicological data related to BDPs are very rare, some studies showed that these polymers have no adverse effect on the environment when degraded (Haider et al., 2019). A previous study tested starch-based biodegradable materials, which are used such as for soil mulching or as polytunnel film for growing strawberries. After a cultivation period of these biodegradable materials buried and degraded in soil, there was no indication of ecotoxicity to the soil and crops (Kapanen et al., 2008). In addition, a study examined Mater-Bi material, which is a biodegradable material made of starch complexed with biodegradable polysters that is used as an agricultural biodegradable film. This study showed that these material degraded completely in the soil and also caused no environmental negative impact (Briassoulis, 2007). Another study on Mater-Bi material to investigate its effect on bacteria, protozoa, algae, plants, earthworms, and crustaceans found that there was no adverse effect of this material on all these tested organisms and Mater-Bi were not toxic to any of them (Sforzini et al., 2016). Moreover, polybutylene succinate (BPS)/starch has been tested for its effect on the bacterial diversity in the soil and on the nitrogen circulation activity in the soil and it was found that there was no adverse effect of the PBS-starch materials (Adhikari et al., 2016). Moreover, a study using polylactic acid (PLA) and PLA/starch blends with various amounts of starch content showed no adverse effect after degradation as tested by the rate of germination and 141 growth of monocotyledon and dicotyledon. Their germination and growth showed no significant difference when compared to control compost with no degraded materials (Rudeekit et al., 2012). Another study on cress and spinach to study the ecotoxicological impact of compost produced from municipal waste and containing polyvinyl alcohol (PVA) biopolymer and PVA + cellulose nanofiber (CNF) nanocomposites under composting conditions showed that there was no negative effect on the germination of the seeds and the plant growth (Salehpour et al., 2018).

Previous experiments (Chapter 3 of this thesis) have tested the degradation of PCL at 55°C (residual remaining) and the results showed that there was significant PCL degradation with time at 55°C but that degradation was significantly different from the PCL degradation at 25°C, 37°C, 45°C and 50°C. The highest percentage of degradation at 55°C was just 49% while it reached 86% at 50°C. This indicates that breakdown products from PCL in powder form are likely to be accumulating in the compost treated at 55°C causing an inhibition of seed germination.

Cress seed germination was further tested in extracts from compost at 55°C with different PCL concentrations to check whether seed germination was inhibited under all PCL concentrations or whether the inhibition was due a certain amount of PCL. It was apparent from these results that 5% PCL concentration in compost at 55°C can still allow some seed germination but with percentage that can only reach 50%. In contrast, lower concentrations of PCL had no effect. It has been stated that 1% loading of biodegradable materials in soil is much higher than the biodegradable plastic amount expected to be in the environment from one application, for example, BDP film for mulching. 1% is considered as the prescribed concentration of plastics needed in order to test the ecotoxicity of any polymer which requires the polymer to be in a very high dose or high concentration. However, the concentration of the biopolymer material that needs to be tested in compost needs to be added at an unrealistically high concentration of 10% while the compost needs to be composted for three weeks (Sforzini et al., 2016).

To investigate the sensitivity of different seeds toward the high concentration of PCL in compost, mustard, lettuce and rocket seeds were tested in extracts from compost at 55°C with 10% PCL. The results of this test showed that again there was no germination for any species of seed tested in compost with 10% degraded PCL. These results confirm that the presence of PCL in the compost with concentration of 5% and above at 55°C can cause 142 seed germination to reduce. However, these results still need further investigations and 10% PCL in compost cannot be considered as toxic because this study did not perform ecotoxicity testing method to test the toxicity of the polymer. Moreover in order to perform ecotoxicity test for any BDP it is important to state the timing for the test not only to have suitable and sensitive assessment because these BDPs can be safe before the degradation but may produce toxic products during the degradation (Degli-Innocenti et al., 2001).

5.6 Conclusion

The main goal of the current chapter was to determine the effect of PCL degradation in compost on seed germination to ensure that the degradation of PCL will not harm the environment and the biota. Cress seeds were used as model seed for this study. This study has found that under most conditions PCL has no adverse effect on the germination of seeds and hence PCL biodegradable polymer can be used for different applications especially agricultural applications without causing any harm or adverse effect to the environment or seed germination. The most important finding of this study was that PCL with concentration of 5% and above at 55°C cannot allow seed germination to occur and hence further investigation is needed in order to test the ecotoxicity of this polymer.

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5.7 References

Briassoulis, D. (2007) ‘Analysis of the mechanical and degradation performances of optimised agricultural biodegradable films.’ Polymer Degradation and Stability, 92(6) pp. 1115–1132.

Degli-Innocenti, F., Bellia, G., Tosin, M., Kapanen, A. and Itävaara, M. (2001) ‘Detection of toxicity released by biodegradable plastics after composting in activated vermiculite.’ Polymer Degradation and Stability, 73(1) pp. 101–106.

Doi, Y., Kanesawa, Y., Tanahashi, N. and Kumagai, Y. (1992) ‘Biodegradation of microbial polyesters in the marine environment.’ Polymer Degradation and Stability, 36(2) pp. 173– 177.

Emadian, S. M., Onay, T. T. and Demirel, B. (2017) ‘Biodegradation of bioplastics in natural environments.’ Waste Management, 59 pp. 526–536.

Ishii, K., Fukui, M. and Takii, S. (2000) ‘Microbial succession during a composting process as evaluated by denaturing gradient gel electrophoresis analysis.’ Journal of Applied Microbiology, 89(5) pp. 768–777.

Kapanen, A., Schettini, E., Vox, G. and Itävaara, M. (2008) ‘Performance and environmental impact of biodegradable films in agriculture: A field study on protected cultivation.’ Journal of Polymers and the Environment, 16(2) pp. 109–122.

Krasowska, K., Heimowska, A. and Morawska, M. (2016) ‘Environmental degradability of polycaprolactone under natural conditions.’ E3S Web of Conferences, (Vol. 10, p. 00048). EDP Sciences.

Kumar, S. and Maiti, P. (2016) ‘Controlled biodegradation of polymers using nanoparticles and its application.’ RSC Advances, 6(72) pp. 67449–67480.

Nair, N. R., Sekhar, V. C., Nampoothiri, K. M. and Pandey, A. (2016) Biodegradation of Biopolymers. In Current Developments in Biotechnology and Bioengineering, pp.739-755. Elsevier.

Qi, X., Ren, Y. and Wang, X. (2017) ‘New advances in the biodegradation of Poly(lactic) acid.’ International Biodeterioration & Biodegradation, 117, February, pp. 215–223.

Rudeekit, Y., Siriyota, P., Intaraksa, P., Chaiwutthinan, P., Tajan, M. and Leejarkpai, T. (2012) ‘Compostability and Ecotoxicity of Poly(lactic acid) and Starch Blends.’ Advanced Materials Research, 506, April, pp. 323–326. 144

Rudnik, E. (2010) Compostable polymer materialse. Elsevier.

Salehpour, S., Jonoobi, M., Ahmadzadeh, M., Siracusa, V., Rafieian, F. and Oksman, K. (2018) ‘Biodegradation and ecotoxicological impact of cellulose nanocomposites in municipal solid waste composting.’ International Journal of Biological Macromolecules, 111, May, pp. 264–270.

Sforzini, S., Oliveri, L., Chinaglia, S. and Viarengo, A. (2016) ‘Application of Biotests for the Determination of Soil Ecotoxicity after Exposure to Biodegradable Plastics.’ Frontiers in Environmental Science, 4, October,p.68.

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Chapter 6

General conclusion and future work

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6.1 Summary

This research investigated the degradation rate of four biodegradable polymers (BDPs) by measuring the weight loss of these polymers under controlled and environmental conditions. It was observed that polycaprolactone (PCL) polymer was the fastest polymer that undergoes degradation under all conditions. The fungi that were responsible for PCL degradation were isolated and tested for their degradability under 50°C temperature conditions (Chapter 2). PCL then underwent several investigations to test its ability to be degraded as different polymer forms (strips and powder) and under different temperature conditions (Chapter 3). Later, the effect of 10% PCL degradation on the fungal community structure in compost was investigated using species marker gene quantification by next generation sequencing (Chapter 4). Finally, the effect of different PCL concentrations on seed germination and seed growth in compost was studied (Chapter 5).

6.2 Chapter 2. A comparative study on the rate of microbial degradation of four biodegradable polymers in soil and compost.

Four different BDPs have been tested for their degradation under controlled laboratory conditions. Polymer discs were buried under soil and compost and incubated at 25°C, 37°C and 50°C (compost only). PCL was found to have the fastest degradation rate among the four types of the tested BDPs at all temperatures. It has been found as well that the degradation rate under compost conditions were more rapid compared to soil conditions and that the degradation rate was greater at higher temperature (50°C). Moreover, the microorganisms at the surface of the polymer discs were isolated and identified and Thermomyces lanuginosus was found to be to most predominant fungus isolated at 50°C from the surface of PCL, PHB and PLA discs. The same BDPs were tested under environmental conditions and only the PCL polymer showed significant mass reduction (P<0.05). Since PCL showed promising results, the study then tested the degradability by T. lanuginosus of PCL in more detail. A degradability test was performed and the results showed that T. lanuginosus was responsible for the degradation of PCL at 50°C and the polymer degradation at this temperature was not due to any physical factors but to biological factors. A previous study had likewise confirmed that a single type of fungus isolated from PCL at 52°C was identified as Thermomyces sp. and they demonstrated that

147 soil temperature had a crucial effect on the polymer degradation and that fungi are the main polymer degraders (Nishide et al., 1999). From this study, it can be confirmed that temperature was a key parameter in the polymer degradation as well as the burial environment where compost was found to accelerate the biodegradation compared to a soil environment. This might be due to the higher organic matter present in the compost which may enhance the presence of microorganisms that will act as polymer degraders. The importance of temperature and compost was seen clearly by the burial of the polymer discs under natural environmental conditions. The burial medium was soil and the temperature never reached 50°C and thus the degradation rate was significantly less than the degradation under controlled conditions. Although PCL degradation showed significant progress under environmental conditions, the degradation never reached 100% even though the incubation period was 21 months in comparison to the 91 days for the complete PCL degradation under controlled conditions.

From all these findings in Chapter 2 it can be concluded that each polymer requires specific environmental conditions for their full degradation, therefore consideration is needed with regard to how the different polymers will be disposed and how the landfill should be managed in order to help prevent the accumulation of these polymers in the environment.

While under controlled conditions three temperatures were used to test the degradation, more temperatures should be included for future study to investigate if there are any differences in the degradation for example between 45°C and 55°C. Moreover, a longer trial period of around 2 or 3 years is needed to determine the long-term fate of the BDPs used in this study. Finally, more focus should take place in the isolation of new thermophilic microorganisms because these will help in composting technology.

6.3 Chapter 3: Characterisation of polycaprolactone as a promising biodegradable polymer

This chapter further quantified PCL degradation after this polymer gave the most promising results from Chapter 2 of this study. PCL polymer was now studied in two different forms: polymer powder and polymer strips. Polymer strips were incubated in compost and compost extract at different temperatures. The findings confirmed that the polymer degradation can be highly

148 affected by temperature, which is considered as the main factor for the PCL degradation. In particular, the tensile strength of the polymer decreased with time and as the temperature increased, and this degradation was mainly due to microbial activity not to hydrolysis because strips incubated in compost extract showed no degradation (Figure 3.1 – 3.4). It is possible that some factors that are present in compost that enhance the polymer degradation are not present in the compost extract. Therefore further analysis is needed to compare the polymer degradation under different conditions, such as sterile compost extract and sterile compost.

Moreover it can be found that the degradation was higher for polymer powder toward the end of the incubation period but it did not reach a complete degradation because the incubation period was too short (eight weeks). A longer incubation period over ten weeks might be required in order to determine whether a complete degradation can be reached. Another future recommendation that should be considered is to isolate and identify the fungi from the surface of the PCL strips and compare them to the fungi present on the surface of the PCL discs (Chapter 2) to give a clearer understanding about the fungal communities present on the surface of the polymers and if the form of the polymer (discs or strips) can affect the fungal community structure.

It was clear as well from this chapter that at 55°C the degradation of the PCL powder were far less compared to the degradation at 50°C. This might be due to less fungal degraders being present at this high temperature compared to the thermophilic fungi that can tolerate 50°C. Moreover, PCL strips cannot be measured at 55°C because they rapidly melt and therefore no data for PCL degradation could be obtained for this temperature. However it might be useful to use the polymer discs at 55°C instead of polymer strips to further examine the degradation characteristics.

6.4 Chapter 4: The impact of polycaprolactone degradation on microbial communities in compost at different temperatures using next generation sequencing

After the promising results showing that PCL can be environmentally friendly and can be degraded in the environment by the action of microorganisms, this warranted more investigation on PCL. Chapter 4 therefore examined the impact of 10% PCL degradation on microbial community structure in compost at different temperatures, through use of

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ITS region sequencing. The work described in this chapter showed that the fungal communities were diverse at 25°C, 37°C, and 45°C both for compost samples with 10% PCL and control samples. However, fungal communities were less diverse at 50°C and 55°C for compost samples with 10% PCL. In addition, the results showed that temperature as well had an influence on fungal community structure beside the presence of PCL and it was apparent that with increasing temperature, the number of operational taxonomic unites (OTUs) decreases. Moreover, under each temperature, principal component analysis (PCA) showed that fungal communities were distributed according to the presence of PCL. However, the capability of PCL to induce changes on the microbial community should be investigated further. Moreover, it is important to highlight that it was challenging to extract DNA from compost samples at 50°C and 55°C which could be a factor in the interpretation of the results at these temperatures and hence further analysis is required.

Many constituents of the fungal community structure might not be necessarily directly involved in the degradation of the polymer (indirect response) but It might be telling more about a broader compost community structure when this polymer present rather than the fungi that is directly driven the degradation. There might be specific fungi that performing degradation and a lot of these have not been classified to species level.

6.5 Chapter 5: The effect of polycaprolactone degradation in compost on seed germination

After investigating the effect of PCL on the fungal microbial community in compost, the final Chapter described the effect of PCL degradation in compost on seed germination. It is very important to look into the effect of any BDPs that will be released into the environment on how its degradation might be affecting the living organisms.

PCL can be used in the composting system because it proved its ability to be degraded under environmental conditions. Degli-Innocenti et al. (2001) have stated that the fate of the biodegradable materials that is released in the environment will end up integrated into the soil. For example, agricultural plastic films will be directly integrated into the soil whereas compostable materials will pass through composting process before entering the soil. In both cases, soil will be the final destination for both materials and therefore it is

150 important to guarantee that these BDPs do not accumulate in the soil or release toxic molecules that can affect living organisms. This study therefore tested seed germination in compost amended with different concentrations of degradable PCL and incubated at different temperatures.

The fast growing cress seeds were used as a model. The degradation of PCL in compost had no adverse effect on seed germination and seedling growth if the compost was at 50°C or below. However at 55°C, PCL extract with a concentration greater than 5% supressed seed germination, and 5% PCL allowed only 50% of seed germination. This seems unclear and might be related to unseen alteration of the structure of PCL at this temperature with high PCL concentration that might cause seed toxicity.

In Chapter 3, it had been shown that while degradation of PCL powder under 55°C was significant, only 50% of PCL was degraded. The rest of PCL might be accumulated in the compost or required more time to be degraded. This also might be due to the low microbial activity at this high temperature. It can be suggested that longer periods for compost incubation at 55°C might be needed before extract from the compost can be used to test seed germination. Moreover, it can be suggested as well that to try germinating the seeds under optimal conditions (in uncontaminated water) and then add the compost extract with 10% PCL after germination to check whether the presence of PCL will supress the growth as well as seed germination.

6.6 General discussion and contribution of this study to the field of knowledge

Testing the degradability of BDPs under different conditions is considered as a key factor that would help future waste management. There are many studies that have investigated the degradation of BDPs (Laycock et al., 2017; Qi et al., 2017; Dilkes-Hoffman et al., 2019). In this study, among four BDPs that have been tested, PCL showed the fastest degradation under controlled laboratory conditions and environmental condition. A number of previous studies have demonstrated rapid degradation of PCL. For example, a study reported that a significant degradation of PCL at 47 days under controlled compost condition at 58°C reached 90% (Funabashi et al., 2007).

It was very clear from this study that the degradation by itself does not matter as much as the suitable conditions that are required for the degradation of each polymer. It was 151 apparent that all the tested polymers are degradable polymers but each requires suitable, specific conditions for the degradation to occur like temperature, pH and moisture. The same results were stated by a recent paper of Haider et al. (2019). In this study temperature seems to be a main factor that driven the degradation of the PCL.

Fungi from the surface of the buried polymers were isolated and identified. Thermomyces lanuginosus was found to be the main degrader of PCL at 50°C in compost conditions. This is considered as the first contribution to the field since this is the first study to the best of our knowledge to isolate T. lanuginosus from the surface of PCL at thermophilic conditions. There are few reports on the degradation of polymers, and none for PCL at high temperatures by fungus (Sanchez et al., 2000). In addition, a recent paper has concluded that most of the PCL principal degraders that have been isolated at high temperatures are bacteria (Emadian et al., 2017).

Moreover a deeper knowledge about microorganisms present in each environment is needed. Therefore next generation sequencing has been used in this study when 10% PCL is present in compost at different temperatures. This is also the first study that quantifies the microbial community structure in compost amended with degraded PCL at different temperatures. PCL has not been previously tested for its effect on microbial community structure. This study found that the number of OTUs decreases with increasing temperature. This might be because fewer microorganisms can present at higher temperatures. A study stated that the number of fungal species that have the ability to survive at temperatures between 45°C and 55°C are few (Maheshwari et al., 2000). On the other hand, fungal community diversity (richness and evenness) is higher at 25°C, 37°C and 45°C at both samples with 10% PCL and control compost compared to compost samples with 10% PCL at 50°C and 55°C. However, diversity indices showed higher diversity in control compost at higher tempertures. Moreover, it was clear that at each temperature, the distribution of the fungal communities was based on the presence of PCL. In addition to that, temperature as well can be considered as a factor that can affect microbial community structures. Nevertheless, the ability of PCL to make changes on the microbial community should be investigated further. Other previous study showed that the biodiversity of microorganisms that can degrade BDPs are differ according to the environmental conditions (Tezuka et al., 2004). Other studies that concern the effect of BDPs on microbial communities are rare and have been discussed earlier in this thesis.

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In addition, more investigations on the effect of PCL on biota have been done and the effect of degraded PCL on seed germination has been studied. Different PCL concentrations at different temperatures have been tested for its toxicity on seed germination and this is another contribution to this field. PCL was found to be safe for the germination of seeds at nearly all temperatures. However, at 55°C the seed germination is inhibited when PCL is present in the compost with concentrations of 5% and above. The reason for this is unclear and further examination is needed.

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6.7 References

Qi, X., Ren, Y. and Wang, X. (2017) ‘New advances in the biodegradation of Poly(lactic) acid.’ International Biodeterioration & Biodegradation, 117, February, pp. 215–223.

Tezuka, Y., Ishii, N., Kasuya, K. and Mitomo, H. (2004) ‘Degradation of poly(ethylene succinate) by mesophilic bacteria.’ Polymer Degradation and Stability, 84(1) pp. 115–121.

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