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Oyster Mushroom) The role of nitrogen sources and caffeine for growth of Pleurotus ostreatus (oyster mushroom) By Claudia Patricia Carrasco Cabrera A thesis submitted in fulfilment of requirements of the degree of Doctor of Philosophy School of Life and Enviromental Sciences Faculty of Science The University of Sydney March 2018 Originality Statement This thesis is submitted to the University of Sydney in fulfilment of the requirement for the Doctor of Philosophy. The work presented in the thesis is, to the best of my knowledge and belief, original except as acknowledged in the text. I hereby declare that I have not submitted this material, either in full or in part, for a degree at this or any other institution. Claudia Patricia Carrasco Cabrera Date: 28. 03. 2018 2 Presentations Part of the research in this thesis has been presented in the following events: Poster at the Symposium Faculty of Agriculture and Environment, The University of Sydney, July 2014. Poster at the 19th Congress of the International Society Mushroom Science, Amsterdam, Netherlands, May-June 2016. Three minute thesis competition (3MT), Faculty of Agriculture and Environment, The University of Sydney, July 2016. 3MT competition, The University of Sydney, August 2016. 3 Abstract Waste products from coffee production are useful substrates for cultivation of Pleurotus ostreatus in coffee-producing countries as this species is relatively easy to grow, substrates are readily available and the mushrooms (or fruiting bodies) are a valuable source of nutrition and income. In developed countries, cultivation P. ostreatus on spent coffee grounds (SCG) from coffee consumption is a novel way to use this form of waste product in urban areas. It is not yet known if high mushroom yields can be achieved by growing P. ostreatus on SCG or if the caffeine content of residues can be substantially reduced by growing mushrooms for safe disposal of coffee waste. In addition, when mushrooms are grown on SCG it is not known how suitable they are for human consumption in terms of caffeine content. Thus, there is a need to understand the role of caffeine during the cultivation of oyster mushroom on SCG as caffeine utilization by this fungus has not yet been described. The study presented here provides key information related to the fate of caffeine during cultivation of P. ostreatus on SCG. A wide range of nitrogen (N) sources (including caffeine) and extracts from fresh and spent coffee grounds were evaluated for their ability to support mycelial growth on solid agar and in liquid culture. Good biomass production was measured for a range of N forms, with inorganic N being the best source for growth. Caffeine also promoted good mycelial growth but only when provided in low concentrations. At higher concentrations, growth was inhibited. 4 Pleurotus ostreatus was cultivated on SCG-amended substrate to evaluate the effect of caffeine during the vegetative and reproductive growth stages. In two trials, P. ostreatus was grown in treatments ranging from pure SCG (SCG100) through to pure sawdust (sawdust100) and with mixtures of these substrates (i.e. SCG75+sawdust25, SCG50+sawdust50 and SCG25+sawdust75). During the vegetative phase of the laboratory- scale study, three of the four treatments became fully colonized (SCG100, SCG25+sawdust75 and SCG50+sawdust50) and of these treatments, only SCG100 and SCG25+sawdust75 developed mushrooms. Caffeine degradation by P. ostreatus occurred when grown on SCG (with and without sawdust) with detection of caffeine and its degradation products in the substrate and fruiting bodies. In a commercial-scale study, full colonization was observed for SCG25+sawdust75 and sawdust100 (control) and SCG50+sawdust50 was almost fully colonized, but fruiting bodies only developed on SCG25+sawdust75 and the control. Again, caffeine degradation occurred when P. ostreatus was grown on treatments including SCG and a decrease in caffeine content of the SCG was found. All of the compounds that have been previously described for fungal degradation of caffeine (xanthine, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, theobromine, paraxanthine, theophylline and caffeine) were detected and identified and a likely degradation pathway was suggested. 5 Acknowledgements I would like to acknowledge my supervisors who have helped me throughout my studies. To Associate Professor Tina Bell, I give my deepest thanks for her time and patience to teach, explain new concepts and interpretation of results. Sincere thanks to Associate Professor Michael Kertesz for guiding me through the experimental and microbiology aspects of my work, as well as integration of results. My thanks to the Chilean scholarship Becas Chile for economical support and to the Faculty of Agriculture and Environment at the University of Sydney for assisting me financially with a Thomas Lawrence Pawlett Scholarship. My special thanks go to Dr Katarzyna Safianowicz who has supported me with both passion about the Fungi Kingdom and with advice in many other aspects. She has become a good friend whose support I treasured very much. I would also like to acknowledge the daily support from my colleague, Dr Cheryl Poon, who always had a piece of advice, and who has also become a great friend. To my friends and colleagues for their support: Dr Kelly Scarlet, Dr Sergio Danaeri, Dr Paola Corneo, Dr Nirmala Liyanage, Dr Malcom Possell, Dr Peri Tobias, Dr Ali Khoddami, Dr Sabastine Ugbaje; and to Niranjan Acharige, Nathan Danckert. Thanks to Ingrid Zamora for your invaluable laboratory assistance. Many thanks to Dr Floris Van Ogtrop for statistical advice, and to Dr Brian Jones for friendly support. I am very grateful to Dr Alex McBratney for professional and personal advice. Thank you to Iona Gyorgy, mi querida Reina, for always being there for me not only for my research requirements but more importantly, for a nice hug when I needed it most and for all your mush-gifts which made many of my days. Thanks to Michael Turner for helping me 6 with sample analysis. Thanks also to Tatjana Matic, Jessica Maley and Carolina Cuenca Cardozo who were always ready to attend to my laboratory needs or to have a chat. Thanks to Mr Graham Price whose company I enjoyed very much while I worked in the Marsh Lawson Mushroom Research Unit. He is always willing to share his knowledge about mushrooms with a cup of tea. Sincere acknowledgments to Dr Noel Arrold who provided the fungal strain used during this reseach, his facilities and technical support during the commercial farm cultivation experiment. His volunteered resources enabled me to do an important part of my research. Thank you to the family Lagos-Boin who “adopted” me and my husband to be part of their family, and with whom we celebrate our traditions. Thank you ‘tios’ for your love and enormous support. To the family Marin-Fuentes for their great support with whom I shared beautiful and cheerful moments. Thanks to the theatre group “Sin Fronteras” for letting me be part of my Latin culture far from home and making precious friendships, and to my Bikram Yoga teachers and fellow yoguis from whom I have learnt so much. Thank you to my ‘amado’ husband Felipe, my dearest friend who followed me to Australia to pursue my dreams. Thank you for picking me up on rainy days and during the scorching Sydney summer, for letting me work or rest at any time, for sharing walks and swims to clear my mind, and for giving up your weekends to just be by my side. Thank you to my family and friends in Chile for their emotional support. Special thanks to my mother Carolina and my twin sister Gisella, even though we were an ocean apart, they were always there for me – Mum, ‘I did it not only because I was curious…I did it for the three of us’. 7 Table of Contents Originality Statement .......................................................................................................................... 2 Presentations ...................................................................................................................................... 3 Abstract ............................................................................................................................................... 4 Acknowledgements ............................................................................................................................ 6 Table of Contents ................................................................................................................................ 8 List of Tables ..................................................................................................................................... 12 List of Figures .................................................................................................................................... 14 List of Abbreviations ......................................................................................................................... 19 Chapter 1 The contrasting roles of naturally-occurring and cultivated fungi ...................................... 21 1.1 Introduction ................................................................................................................................ 21 1.2 Fungi in natural and managed ecosystems ................................................................................ 21 1.3 Exploitation of fungi .................................................................................................................... 26 1.3.1 Fungi as a food source ........................................................................................................
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