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Identification and Classification of Marine Bacteria Associated with Poly(Ethylene Terephthalate) Degradation

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Identification and classification of marine bacteria associated with poly(ethylene terephthalate) degradation Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy by Hooi Jun Ng Department of Chemistry and Biotechnology School of Science Faculty of Science, Engineering and Technology Swinburne University of Technology November 2014 Abstract Poly(ethylene terephthalate) (PET) is manmade synthetic polymer that has been widely used over the past few decades due to its low manufacturing cost, together with desirable properties. The high production and usage of PET, together with the inappropriate handling of resultant wastes are becoming a major global environmental issue, especially in the marine environment due to the fact that PET is fairly stable and not easily degraded in the environment. The waste handling methods that are currently available, such as burying, incineration and recycling, have their own drawbacks and limitations. Biodegradation represents an environmentally friendly, cost effective and potentially more efficient method for the management of PET, as can be concluded historical instances where microorganisms have been shown to be capable remediating and biodegrading environmental pollutants. The potential with which microorganisms could adapt to mineralize PET has previously been reported, however, none of the studies have identified the potential of marine bacteria to biodegrade of PET. In this project, a collection of marine bacteria belonging to two phylotypes, Alpha- and Gammaproteobacteria, which might have the potential to biodegrade PET, have been investigated to examine their ability to degrade PET. One strain, affiliated to the genus Marinobacter, and designated as A3d10T, was identified to have the ability to hydrolyze bis(benzoyloxyethyl) terephthalate, a trimer of PET. Further investigation of a short term biodegradation experiment also indicated that PET films exposed to this strain underwent significant changes with regard to their surface nanostructure, including increased crystallinity and hydrophilicity, as confirmed by surface topography, surface chemical composition analysis, and wettability, using AFM, Raman spectroscopy, and goniometry, respectively. Representative strains of two genera, which were included in the investigation of PET degradation ability, namely Alteromonas and Marinobacter, were subjected to detail taxonomic investigation. Comparative phylogenetic and genomic analyses, together with traditional physiological and biochemical studies coupled with newly developed modern taxonomic tools, namely multi-locus sequence analysis (MLSA) and MALDI-TOF mass I spectrometry, allowed formal description of a new species of the genus Alteromonas, Alteromonas australica (= type strain H17T = KMM 6016T = CIP 109921T), and two new species of the genus Marinobacter, Marinobacter similis (= type strain A3d10T = JCM 19398T = CIP 110589T = KMM 7501T) and Marinobacter salarius (= type strain R9SW1T = LMG 27497T = JCM 19399T = CIP 110588T = KMM 7502T). The whole genomes of the two newly proposed Marinobacter species were assembled and deposited in public databases, in which the whole genome of M. similis A3d10T provides an opportunity to investigate the genes/enzymes responsible for PET degradation. The development, evaluation and application of modern taxonomic tools such as multi-locus sequence analysis (MLSA) and MALDI-TOF mass spectrometry in the taxonomy of marine bacteria of the genus Alteromonas, and genome taxonomic parameters in the taxonomy of the genus Marinobacter provided alternative and/or complementary path for the description of a new species, making a significant contributing to the analytical techniques that can be used in modern systematic classification of bacteria. II Acknowledgements First and foremost, I would like to thank my principal supervisor, Professor Elena Ivanova for taking me on this project in the second year of my PhD candidature. Her dedicated supervision and guidance throughout this project, as well as her support and time sacrificed during the production of manuscripts, papers, and this thesis is invaluable. Similarly, to my co-supervisor, Professor Russell Crawford and Dr. François Malherbe, thank you for providing feedback on my manuscripts, papers and thesis. Without your support, some of this task would not have been completed easily. I would also like to thank Dr. John Fecondo and Associate Professor John Patterson for their supervision and guidance during the first and second year of my PhD. I am grateful to have the opportunity to work with you both. To all the technical staff and my lab mates, your helps, suggestions, support and company have made my experience in the lab memorable. Big thanks to Ngan who provided constructive advice on microbiology related work, and Soula, Chris, Huimei, Nina and Andrea for their technical supports given during the course of this study. My heartfelt appreciation goes to all co-workers in Prof. Ivanova group for their unreserved support and guidance in a way or another. To Dr. Hayden Webb, thank you for assisting in countless individual experiments and analyses, in particular the surface analysis experiment. To Ha and Chris, thank you for your assistance in performing SEM for the characterization of Marinobacter species. To collaborators, my sincere acknowledgement goes to Dr. Henry Butt, Rachel Knight and all the staff at Bioscreen Medical laboratory for allowing me to use their MALDI-TOF mass spectrometer. I am really happy to have met you guys and thanks for your friendliness that have always made me feel welcome in your lab. I would also like to thank Dr. Nicholas Williamson at Bio21 Institute for his assistance in the use of MALDI- TOF mass spectrometry. Also, I would like to acknowledge the kindness of Prof Georg M. Gǖbitz from Graz University of Technology, Austria for providing 3PET powder, and III Professor Tomoo Sawabe from Hokkaido University, Japan for facilitating the sequencing of the whole genome sequences of strain A3d10T and R9SW1T. To all the academic staff at Department of Chemistry and Biotechnology, thank you for your direct or indirect support, especially during the hard time when my previous supervisor departed. Special thanks go to Dr. Daniel Eldridge, Dr. Tony Barton, and Professor Linda Blackall for giving me the opportunity to involve in teaching. It has been a pleasure to work with you, and thank you for sharing your teaching experience with me. I gratefully acknowledge Swinburne Research for providing me scholarship to pursue my PhD study. Also, thanks to all the staff in the research office for providing me support and guidance in candidature related issue. Finally, I would like to thank my parents for their financial support and understanding throughout my study. To Jiawey, thank you for your care, constant help, support and company throughout the years. IV Declaration I hereby declare that this thesis is my original work and, to the best of my knowledge, this thesis contains no material previously published or written by another person, except where due reference is made in the text. None of this work has been submitted for the award of any other degree at any university. Wherever contributions of others were involved every effort has been made to acknowledge the contributions of the respective workers or authors. Hooi Jun Ng November, 2014 V List of Publications Book Chapters: Webb, H.K., Ng, H.J., Ivanova, E.P. (2014) The Family Methylocystaceae. The Prokaryotes – Alphaproteobacteria and Betaproteobacteria. Springer-Verlag Berlin Heidelberg, p. 341-347. Ivanova, E.P., Ng, H.J., Webb, H.K. (2014) The Family Pseudoalteromonadaceae. The Prokaryotes – Gammaproteobacteria. Springer-Verlag Berlin Heidelberg, p. 575-582. Peer-reviewed journal articles: Ng, H. J., López-Pérez, M., Webb, H. K., Gomez, D., Sawabe, T., Ryan, J., Vyssotski, M., Bizet, C., Malherbe, F., Mikhailov, V. V., Crawford, R. J., Ivanova, E. P. (2014) Marinobacter salarius sp. nov. and Marinobacter similis sp. nov., isolated from sea water. PLoS ONE 9(9): e106514. Ng, H.J., Webb, H.K., Crawford, R.J., Malherbe, F., Butt, H., Knight, R., Mikhailov, V.V., Ivanova, E.P. (2013) Updating the taxonomic toolbox: classification of Alteromonas spp. using multilocus phylogenetic analysis and MALDI-TOF mass spectrometry. Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology, 103 (2), p. 265-275. Ivanova, E.P., Ng, H.J., Webb, H.K., Feng, G., Oshima, K., Hattori, M., Ohkuma, M., Sergeev, A.F., Mikhailov, V.V., Crawford, R.J., Sawabe, T. (2014) Draft genome sequences of Marinobacter similis A3d10T and Marinobacter salarius R9SW1T. Genome announcements, 2(3):e00442-14. Ivanova, E.P., Ng, H.J., Webb, H.K., Kurilenko, V.V., Zhukova, N.V., Mikhailov, V.V., Ponamareva, O.N., Crawford, R.J. (2013) Alteromonas australica sp. nov., isolated from Tasman Sea. Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology, 103 (4), p. 877-884. VI Conferences with published abstracts: Ng, H. J., Webb, H. K., Crawford, R. J., Malherbe, F., Patterson, J., Ivanova, E. P. (2011). Alteromonas australica sp. nov., a poly(ethylene terephthalate) degrading bacterium, isolated from the Tasman Sea, Pacific Ocean, Victoria, Australia. Australian Society for Microbiology Annual Meeting. Hobart, Tasmania. Ng, H. J., Webb, H. K., Malherbe, F., Patterson, J., Ivanova, E. P. (2011). Development of multilocus sequence analysis (MLSA) and MALDI-TOF mass spectrometry for Alteromonas
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