Microbial cell attachment, colonisation and degradation of coal Nur Hazlin Hazrin Chong A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science The University of New South Wales Sydney, Australia September 2013 Abstract Discoveries of aerobic coal degrading microorganisms have led to their utilisation in various biotechnological coal processes. One promising application of these microbes is the acceleration of coal to methane, which provides an avenue for more sustainable coal usage. However, despite the various findings of coal degraders and related mechanisms, a key aspect in coal degradation, which is cell attachment and colonisation, has been largely neglected. This study is among the first to describe in detail microbial cell attachment and colonisation on coal. Using coal-degrading bacteria and fungi, the initial cell attachment and biofilm formation on coal were investigated across different coal types and conditions. Physico-chemical analyses based on contact angle measurements revealed that hydrophobicity, surface free energy and adhesion thermodynamics, as well as secondary biological and environmental factors, played a crucial role in governing the first form of cell interaction with coal. Direct observation and electron microscopy highlighted different colonisation mechanisms on coal based on cell morphology, surface topography and environmental conditions. Correlations were found between colonisation and degradation of coal, which stressed the importance of cell attachment in coal degradation, although exceptions were present. Another interest of this study was to isolate native coal-degrading fungi for potential field applications. Through multiple coal degradation screenings, Fusarium oxysporum G9o was discovered as a promising bituminous coal-degrading fungus. The isolate showed softening of raw bituminous coal, and infrared analyses revealed oxidation and cleaving mechanisms of coal components. Further, the colonisation of coal by soil communities was monitored through microbial community analyses using Terminal- Restriction Fragment Length Polymorphism (T-RFLP) and pyrosequencing analyses. Unique communities from soil were identified as dominant colonisers on coal, which have not been previously revealed through conventional cultural techniques. Overall, the findings in this study provide valuable insights into the mechanisms of cell attachment and colonisation on coal. This serves as a foundation for a new research area in coal microbiology, which will increase our currently limited understanding on coal-cell interactions. ii Dedication This thesis is dedicated to my four pillars of life, God, my parents, and husband God, for creating my existence in life, for providing me sustenance, for showing me strength and patience, and for showering your Love and Mercy Mum and Dad, for being the reason I am here today, for your unconditional and constant love and support, and for my valued and healthy upbringing My husband, for being my best friend, for sharing my every happiness and sadness, and for your endless love and support iii Acknowledgements I am eternally grateful to have met and worked with an amazing group of people, of whom without their help, this thesis could not have been written. Thank you to my supervisor Dr Mike Manefield for his excellent guidance, support and encouragement throughout my whole candidature. Mike, you never cease to inspire me to become a better scientist, thinker and writer. Your words and gestures have made a positive impact to me as a young researcher and I am forever thankful to have embarked on this PhD journey with you as my guide. Thank you for believing in me. I hope I have made you proud and will continue to do so in my future career. Thank you to Dr Maria Luisa Gutierrez-Zamora for her dedicated support, guidance and friendship. I truly appreciate all your help and advice on so many aspects of this PhD project, right from when I first started until I handed in my thesis. I owe you a lot for teaching me the bulk of the molecular work done in this study. Your positive vibe in the lab and our meetings were always infectious to me and everyone else. I am so thankful to know you as an excellent researcher and friend! Special thanks to Dr Chris Marjo and Dr Anne Rich from the Solid State Elemental Analysis Unit at UNSW for their kind help and support in teaching me everything about FTIR. Thanks to Anne for showing me how to prepare the coal samples, and operate and troubleshoot the FTIR machine. Thanks to Chris for his thorough explanations on interpreting FTIR spectra and the complexity (and discovered beauty) behind it. Thank you to Jenny Norman from the Electron Microscopy unit at UNSW for the training and technical advice to produce high quality SEM micrographs, which were necessary for fundamental observations on the bacterial and fungal attachment on coal. I am thankful to Dr Theerthankar Das for his expert help and excellent support in the thermodynamics of adhesion works in this study, which played an important role in the discovery on the physico-chemical relationship between microbes and coal. Thank you to Professor Colin Ward for his geological advice on coal. Especially thank you for providing me with the lignite coal, which gave very useful results in this study. Also thanks to Joanne Wilde from the BEES Rock Preparatory lab at UNSW for her coal polishing services. iv Thanks very much to Shaun Nielsen for teaching me how T-RFLP and pyrosequencing work, and Dr Ezequiel Marzinelli for teaching me statistical analyses using the PRIMER-E software. Thanks to John Webster for his advice on sub-sampling my pyrosequencing data. Thank you to Dr Sohail Siddiqui and Dr Haluk Ertan from the Cavicchioli lab for kindly showing me how to perform the enzymes assay and calculations. To the Manefield group, I am grateful to have you as my research mates. Mona, Iman, Onder, Das, Sharma, Leena, Matt, Valentina, John and Sabrina- you guys are a terrific bunch. Thank you for all your help in the lab and office and for the nice and/or funny conversations we’ve had. Special thanks to Dr Adrian Low for all your help, and Mona El Hassan and Iman Taleb for your great friendship. I want to thank Professor Staffan Kjelleberg and Professor Peter Steinberg from the Centre for Marine Bioinnovation for allowing me to be a part of their fantastic research group. Special thanks to Kirsty, Leena, Adam, Chau and Amy for their helpful administrative support. Thanks Gee, Raymond, Vipra and the whole 304/616 lab for your help and support whenever I needed them. Not forgetting Arthur Bauer, Josh Glass and Sophie Holland as wonderful and helpful interns! Thank you to Peter Francis and Glen Cunningham from Biogas Energy Pty. Ltd. for funding my research. A special thanks to the coal to methane (C2M) group led by Mike and Dr Torsten Thomas for giving me helpful feedback in the meetings. Thank you to the Malaysian Ministry of Higher Education and National University of Malaysia for financially supporting my study abroad and ensuring my well being as an international student. Special thanks to James Hayton for giving me helpful advice and support throughout the final months of my PhD. It’s been great working with you! Thank you to all my lovely akhawat: Kak Mel, Kak Hani, Afida, Ainul, Dayah R, Ain, Elyna, Nadiah and the rest (it’s a long list!) for being there for me and supporting through my PhD journey. Uhibukkunna fillah abadan abada. Last, but definitely not least, a HUGE thank you and appreciation to Arif for being the most loving, understanding, supportive and patient husband I can ever have. Your constant push and motivation for me to do my best are deeply appreciated. You are truly a kind person and I am grateful to have the best partner in life. Thank you Mama and Baba, for raising Arif to become a wonderful person he is today. v Table of contents Abstract ............................................................................................................... ii Dedication .......................................................................................................... iii Acknowledgements ............................................................................................ iv Table of contents ............................................................................................... vi List of publications .............................................................................................. x List of figures ...................................................................................................... xi List of tables ...................................................................................................... xv Abbreviations ................................................................................................... xvi 1 A literature review on the microbial cell attachment and degradation of coal ..................................................................................................................... 1 1.1 Overview ...........................................................................................................1 1.2 Coal ....................................................................................................................4 1.2.1 Origin of coal ...............................................................................................4 1.2.2 Coal ranks ...................................................................................................4