BIODEGRADATION OF POLYURETHANE UNDER COMPOSTING CONDITIONS A thesis submitted to The University of Manchester for the degree of DOCTOR OF PHILOSOPHY in the Faculty of Life Sciences 2013 Urooj Zafar Table of Content Table of Content ................................................................................................................ 2 List of Figures ................................................................................................................... 6 Chapter 1 .................................................................................................................................................. 6 Chapter 2 .................................................................................................................................................. 6 Chapter 2 Appendix ............................................................................................................................. 6 Chapter 3 .................................................................................................................................................. 7 Chapter 3 Appendix ............................................................................................................................. 7 Chapter 4 .................................................................................................................................................. 7 Chapter 4 Appendix ............................................................................................................................. 7 List of Tables..................................................................................................................... 8 Chapter 1 .................................................................................................................................................. 8 Chapter 2 .................................................................................................................................................. 8 Chapter 2 Appendix ............................................................................................................................. 8 Chapter 3 .................................................................................................................................................. 8 Chapter 3 Appendix ............................................................................................................................. 9 Chapter 4 .................................................................................................................................................. 9 Chapter 4 Appendix ............................................................................................................................. 9 Abstract ........................................................................................................................... 10 Declaration ...................................................................................................................... 11 Copyright statement ........................................................................................................ 11 List of Abbreviation ........................................................................................................ 12 Acknowledgement........................................................................................................... 14 Chapter 1 ......................................................................................................................... 16 1. Introduction ................................................................................................................. 16 1.1 Types of plastics ............................................................................................................................... 18 1.2 General mechanisms of degradation of plastic polymers ............................................... 18 1.2. 1 Photo degradation ................................................................................................................. 19 1.2. 2 Thermal degradation............................................................................................................ 19 1.2. 3 Ozone induced degradation .............................................................................................. 19 1.2.4 Mechano-chemical degradation........................................................................................ 20 1.2.5 Biodegradation ........................................................................................................................ 20 1.3 Biodegradable plastics................................................................................................................... 21 1.4 Polyurethanes ................................................................................................................................... 22 1.4.1 Chemical structure ................................................................................................................. 23 1.4.2 Types of polyurethanes ........................................................................................................ 24 1.4.3 Biodegradation of polyurethanes .................................................................................... 27 1.5 Composting ........................................................................................................................................ 34 1.5.1 Bacteria associated with compost ................................................................................... 41 1.5.2 Fungi associated with compost ......................................................................................... 42 1.5.3 Actinomycetes associated with compost ...................................................................... 43 2 1.5.4 Biodegradation under composting conditions ........................................................... 44 1. 6 Techniques used to study microbial ecology ...................................................................... 45 1.6.1 Molecular approaches to studying microbial diversity .......................................... 45 1.6.2 Techniques based on PCR amplification of microbial community DNA/ PCR dependent techniques...................................................................................................................... 46 1.6.2. 1 Cloning and characterization ........................................................................................ 47 1.6.2.4 Terminal Restriction Fragment Length Polymorphism (TRFLP) ................... 53 1.6.2.6 454 pyrosequencing........................................................................................................... 57 1.6.3 Community diversity analysis ........................................................................................... 60 1. 8 Summary ............................................................................................................................................ 62 1. 9 Experimental aims .................................................................................................... 62 1.10 Alternative format ........................................................................................................................ 64 1.11 References ............................................................................................................... 66 Chapter 2: Fungal community associated with biodegradation of polyester polyurethane buried under compost at different temperatures ........................................ 80 2.2 Introduction ............................................................................................................... 83 2.3 Materials and Methods .............................................................................................. 85 2.3.1 Media composition ...................................................................................................................... 85 2.3.1.1 R2A ............................................................................................................................................. 85 2.3.1.2 Potato dextrose Agar ......................................................................................................... 85 2.3.1.3 Compost Extract .................................................................................................................. 85 2.3.1.4 Polyurethane agar (PUA) ................................................................................................. 86 2.3.2 Fabrication of polyurethane coupons .................................................................................. 86 2.3.3 Burial of polyurethane coupons in compost ..................................................................... 87 2.3.4 Recovery of polyurethane samples and enumeration of microbes ......................... 88 2.3.5 Microscopic analysis of PU coupons .................................................................................... 89 2.3.6 Tensile strength and weight loss determination ............................................................ 89 2.3.7 Extraction, amplification and purification of genomic DNA from the isolated PU degrading fungal colonies .................................................................................................................... 90 2.3.8 ITS rDNA sequencing and identification ............................................................................ 91 2.3.9 Extraction and amplification