Biodegradation of poly(ethylene terephthalate) by marine bacteria, and strategies for its enhancement Submitted in total fulfillment of the requirements for the degree of Doctor of Philosophy by Hayden Webb Environmental and Biotechnology Centre Faculty of Life and Social Sciences Swinburne University of Technology May 2012 ___________________________________________________________________ Abstract Plastic accumulation, particularly in the world’s oceans is of increasing environmental concern. One of the major components of plastic waste is poly(ethylene terephthalate) (PET), a polymer frequently used in many applications, including textiles and food packaging. The current methods of disposal of PET waste, landfill, incineration and recycling, each have inherent drawbacks and limitations, and as such there is a need for efficient and cost-effective alternative. Biodegradation is an attractive option for environmentally friendly and efficient disposal of plastic waste. To date, no protocol has yet been developed to feasibly dispose of PET by biodegradation con a commercial scale. The current works aims to investigate the potential of PET biodegradation as a plastic disposal procedure by providing fundamental knowledge of biodegradation processes, and to develop strategies for improving biodegradation efficiency. PET samples were incubated in marine bacterial community enrichment cultures, and the dynamics of the polymer – bacterial interactions traced. Modifications to polymer surfaces were monitored using a variety of surface characterisation techniques, including atomic force microscopy (AFM), x-ray photoelectron spectroscopy (XPS) and infrared microspectroscopy using Synchrotron radiation. Taxonomic members of the bacterial enrichment cultures that developed in the presence of PET were recovered and identified via 16S rRNA gene sequencing. Marine bacteria were shown to possess the ability to degrade PET surfaces. Larger topographical surface features were degraded preferentially, which resulted in overall smoothening of the polymer surface. Subtle changes in the chemistry of the PET surface were also detected, which were indications of polymer chain cleavage and oligomer excision. Bacterial enrichment communities were dominated by members of α- and γ- proteobacteria, with contributions from β-proteobacteria. Among the isolates with the highest potential for PET biodegradation, many returned nearest taxonomic relatives of species of Thalassospira, Kordiimonas and Alteromonas. Commensal relationships i appeared to be required for effective PET biodegradation, with several sulphur-cycling strains showing potential participation in PET degrading communities. Several strategies for enhancement of the rate of PET biodegradation were also put forward. Increasing the size of bacterial enrichment communities by supplementation with essential elemental nutrients in nitrogen and phosphorus proved effective in increasing the rate of biodegradation, as did alteration of the composition of the communities by increasing the selective advantage of PET-degrading strains through incubation in dark conditions. An alternative approach through manipulation of the polymer was another effective option; pre-treatment of PET samples with surfactants increased their surface roughness, which enabled bacterial cells greater access to the polymer chains. Limited cell attachment to PET surface was identified as one of the major barriers to biodegradation. To address this, several unrelated surface topographies were tested for the ability to promote cell adhesion, for eventual application on PET surfaces. Of those tested, a superhydrophobic topography based on the structure of the lotus leaf showed the most promise for enhancement of biodegradation through promotion of cell attachment. The results of this study represent a significant contribution to the fundamental understanding of PET biodegradation mechanisms. The knowledge obtained here can be used for the further development of biodegradation as a technique for disposal of PET waste. ii Acknowledgements First and foremost, I would like to acknowledge and thank my primary supervisor, Professor Elena Ivanova for all her guidance and support over the course of this study. The inspiration, motivation and encouragement she provided was a key driving force behind the project, and I am particularly grateful that she (almost) always was able to take time out of her busy schedule to talk things over whenever I needed advice. Similarly, I would also like to thank Professor Russell Crawford for co-supervising the project, and for providing feedback on the various manuscripts and papers that I prepared. His support and encouragement were vital for completion of the work. To Vi Khanh Truong and Jafar Hasan, thank you for your co-operative efforts on countless individual experiments and analyses; we make a pretty good team. I value your friendship, and I’ll always remember the ‘deep philosophical discussions’ we had on a daily basis. I’d like to express my thanks to Hooi Jun Ng, The Hong Phong Nguyen and Rebecca Flecknoe for their assistance in cultivating, counting and characterising bacterial strains. You guys really made my life a little easier. Thank you to Dr. James Wang for his assistance in performing SEM and in early AFM experiments, without his expertise I would have been lost. Also to Dr. Natasa Mitik-Dineva for lighting the way with her earlier work on bacterial attachment. A special thank you goes to Chris, Soula, Ngan, Savithri and all the laboratory technicians who have helped me throughout the course of this work. To all my friends and colleagues at Swinburne for making my time here enjoyable and rewarding. You guys made my time here worthwhile, and although I can’t thank you all individually I would like to specifically thank Abdullah, Bita, Elisa, Fiona, Kaylass, Liz, Pete and Sarah. And finally thank you to my family. It goes without saying, but I’ll say it here: your love and support have shaped me into the person I am today. iii Declaration I, Hayden Webb, declare that this thesis is my original work and contains no material that has been accepted for the award of Doctor of Philosophy, or any other degree or diploma, except where due reference is made. I declare that to the best of my knowledge this thesis contains no material previously published or written by any other person except where due reference is made. Wherever contributions of others were involved every effort has been made to acknowledge the contributions of the respective workers or authors. Signature ______________________________________________________________ iv List of publications Online Book: Webb, H. K., Truong, V. K., Jones, R. T., Crawford, R. J. & Ivanova, E. P. (2010). X- ray photoelectron spectroscopy as a tool in the study of nanostructured titanium and commercial PET surfaces in biotechnological applications. (Nova Science Publishers Inc., New York). Book Chapters: Webb, H. K., Truong, V. K., Jones, R. T., Crawford, R. J. & Ivanova, E. P. (2010). X- ray photoelectron spectroscopy as a tool in the study of nanostructured titanium and commercial PET surfaces in biotechnological applications. In: Wagner, J. M. X-ray Photoelectron Spectroscopy (Nova Science Publishers Inc., New York). Peer-reviewed articles: Ivanova, E. P., Truong, V. K., Webb, H. K., Baulin, V. A., Wang, J. Y., Mohammodi, N., Wang, F., Fluke, C. & Crawford, R. J. (2011). Differential attraction and repulsion of Staphylococcus aureus and Pseudomonas aeruginosa on molecularly smooth titanium films. Scientific Reports 1: 165-172. Webb, H. K., Truong, V. K., Hasan, J., Fluke, C., Crawford, R. J. & Ivanova, E. P. (2011). Roughness parameters for standard description of surface nanoarchitecture. Scanning [In Press]. Webb, H. K., Hasan, J. Truong, V. K., Crawford, R. J. & Ivanova, E. P. (2011). Nature inspired structured surfaces for biomedical applications. Current Medicinal Chemistry 18: 3367-3375. Webb, H. K., Truong, V. K., Hasan, J., Crawford, R. J. & Ivanova, E. P. (2011). Physico-mechanical characterisation of cells using atomic force microscopy - current research and methodologies. Journal of Microbiological Methods 86: 131-139. Ivanova, E. P., Webb, H., Christen, R., Zhukova, N. V., Kurilenko, V. V., Kalinovskaya, N. I. & Crawford, R. J. (2010). Celeribacter neptunius gen. nov. v sp. nov., a new member of Alphaproteobacteria. International Journal of Systematic and Evolutionary Microbiology 60: 1620-1625. Webb, H. K., Crawford, R. J., Sawabe, T. & Ivanova, E. P. (2009). Poly(ethylene terephthalate) polymer surfaces as a substrate for bacterial attachment and biofilm formation. Microbes and Environments 24: 39-42. Peer-reviewed conference proceedings: Webb, H. K., Crawford, R. J., and Ivanova, E. P. (2010). Bacterial modifications of the surface chemistry and nanotopography of poly(ethylene terephthalate). International Conference on Nanoscience and Nanotechnology. Sydney, NSW. Conference presentations with published abstracts: Webb, H. K., Crawford, R. J., and Ivanova, E. P. (2010). Biodegradation of poly(ethylene terephthalate) plastic by marine bacteria. Australian Society for Microbiology Annual Meeting. Sydney, NSW. Webb, H. K., Crawford, R. J., and Ivanova, E. P. (2009). Characterisation of two dominating bacterial phylotypes associated with poly(ethylene terephthalate) polymer degradation. Australian Society for Microbiology Annual Meeting. Perth, WA. Conference posters with published abstracts: Webb, H. K., Crawford, R. J., Ivanova,