Mechanisms of Microbial Methane Production from Sub-Bituminous Australian Coal John Webster A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Science Faculty of Science August 2015 Originality Statement ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed....................................... Date.......................................... COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed ……………………………………………........................... Date ……………………………………………........................... AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ……………………………………………........................... Date ……………………………………………........................... Acknowledgements First and Foremost, I would like to express my gratitude to Torsten Thomas and Mike Manefield for accepting me as their student and for their guidance throughout my PhD program. The opportunity that they provided for me is most appreciated and it looks like it paid off in the end (See following six chapters). I would also like to thank my mum Susan Webster, dad David Webster and sister Elyssa Webster for sticking by me throughout this trying time. Thank you for your support because without the support and love of my family, this achievement would not mean as much to me. To my grandma Helen who was telling people I was a doctor well before the conferral of my degree, I thank you for your support and excitement also and you may now legitimately tell people I am a doctor. Thank you to Leena and Malu who were part of the project and provided both an experienced view to academic dynamics and a sympathetic ear when things got rough. The pressure and stresses on PhD students are immense and its individuals such as you that can make the difference between a good day and a bad day. I would also like to thank my various lab pals, though numerous, I would like to make sure you are all mentioned as you all played a profound role in my life the last few years. So a big thank you to Enrique, Alexandra, Shaun, Mary, Rajesh, Jim, Manue, Lucas, Ricardo and Valentina as well as the international friends we made in Chris Johansson, Michael Roggenbuck and Jenny Staudigl. I’d also like to specifically thank David Reynolds for helping keep the lab organised, interesting and MagicTM. Though we may have wasted quite a bit of time, it was a welcome and well needed reprieve from the daily PhD grind. As well as Ana (not the spider) Esteves for her great sense of humour and for providing the short and feisty demographic our lab was missing. A great friend who I know I can always count on and plus, she’s also pretty smart. I Though a major part of my life was based around university, support from friends outside this environment is also important and a major thank you has to go out to my buddies in crime, Emily Funnell and Rachel Siskovic, my oldest (by only a day) and dearest friend James Tawadros and also the second love of my life Jared Yan (Platonically of course). And most importantly I thank the first (sorry Jared) love of my life, my partner and my best friend, Ashlea Grewar. These years have been trying, but you have always been my constant and my rock. Without your support and love, I can’t imagine where I would be. I’m so glad we have been able to take this journey together and I couldn’t think of anyone else I would want at my side. "I don't give a rat's ass whether it's science or magical power. No, I guess if I had to choose, I'd rather put my money on the power of science. Humans who used to only roam around on the ground are able to fly now! And finally, we're about to go into outer space. Science is a "Power" created and developed by humans. And science just might be what saves this planet. I was able to earn my living thanks to science. So to me, there's nothing greater!" -Cid Highwind II Table of Contents Acknowledgements .................................................................................................................................. I Table of Contents ................................................................................................................................... III Table of Figures ..................................................................................................................................... VII Table of Tables ........................................................................................................................................ X Abstract .................................................................................................................................................. XI Chapter One: Introduction ...................................................................................................................... 1 What is coal? ................................................................................................................................... 1 Its Structure: ................................................................................................................................... 2 Australian Coal Deposits: ................................................................................................................ 3 Coal as a Fuel: ................................................................................................................................. 4 Breakdown of Coal: ......................................................................................................................... 5 Uses of Methane: ............................................................................................................................ 6 Methanogens: ..................................................................................................................................... 7 Degradation of Coal Hydrocarbons: .............................................................................................. 11 Microbial Communities and Methane Production from Coal: ......................................................... 15 Chapter Two: Chemical Characterisation of Soluble Compounds in Australian Coal ........................... 18 Introduction: ..................................................................................................................................... 18 Material and Methods: ..................................................................................................................... 27 Coal Sampling Locations: .............................................................................................................. 27 Coal characteristics ....................................................................................................................... 29 Coal Preparation for chemical analysis: ........................................................................................ 29 Small Apolar Chemical Compound Extraction: ............................................................................. 29 Gas chromatography Flame Ionisation Detection (GC-FID) of DCM extractions: ......................... 30 Alignment and Analysis of GC Chromatogram Peaks: .................................................................. 30 Analysis of extractions with GC-MS: ............................................................................................. 31 Oxidative chemical and enzymatic treatments to coal: ................................................................ 32 Aqueous Extract Analysis: ............................................................................................................. 32 Results: .............................................................................................................................................