Streptomyces as a Source of Geosmin and 2-methylisoborneol Associated Taste and Odour Episodes in Drinking Water Reservoirs Elise Anne Asquith BEnvScMgt (Hons) A thesis submitted to the University of Newcastle, Australia, in fulfilment of requirements for admission to the degree of Doctor of Philosophy February 2015 1 DECLARATION The thesis contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to the final version of my thesis being made available worldwide when deposited in the University’s Digital Repository, subject to the provisions of the Copyright Act 1968. ………………………………….. Elise Anne Asquith I ACKNOWLEDGEMENTS There are a number of individuals who have been of immense support during my PhD candidature who I wish to acknowledge. It has been a challenging and enduring experience, but the end result has to be recognised as a great sense of academic achievement and personal gratification. I would like to express my deep appreciation and gratitude to my supervisors. Dr Craig Evans has undoubtedly been the most important person guiding my research over the past three years and has been a tremendous mentor for me. I am truly grateful for his advice, patience and support. In particular, I wish to thank him for accompanying me on all of my visits to Grahamstown and Chichester Reservoirs and generously dedicating much time to reviewing my thesis. Thanks also goes to my other supervisors. To Associate Professor Phillip Geary for his support and assistance in the revision of the thesis and Professor Hugh Dunstan who provided brilliant and novel ideas which have been of immense importance in directing much of this research. Hunter Water Corporation provided the financial support for conducting this research and I especially would like to thank Mr Bruce Cole for proposing the interesting research topic of this thesis. I also would like to acknowledge The University of Newcastle, Australia, for providing me with a Postgraduate Research Scholarship. I wish to thank all the staff and students associated with the Dunstan-Roberts Laboratory. Dr Margaret MacDonald for her continuous encouragement, Mr Tony Rothkirch for his technical assistance and Nicole, Marcus, and Mousa for their friendship and support during my PhD. I am also very appreciative of the generosity of researchers from the Reproductive Science Laboratory, especially Dr Matthew Jobbling and Dr Ben Curry for their assistance in the molecular analyses conducted in this research. Luke, my loving husband, needs to be recognised as the most significant individual who has been patient and understanding, has supported me and given me continuous encouragement to complete my PhD. I also would like to acknowledge the important role of my parents, Angela and Ralph in getting me to this level of my education. They have given me every opportunity in life and have always encouraged me to reach my fullest potential. Words cannot express how grateful I am to them. Of course my siblings Nivi, Melissa and Joe deserve a mention and I thank them for pretending to show interest in my research over the years. Thanks goes to my canine companion Cooper who has sat loyally and patiently by my side during the writing of this thesis, keeping me company and taking me for much needed runs to clear my mind. II “An understanding of the natural world and what's in it is a source of not only a great curiosity, but great fulfillment” Sir David Attenborough III TABLE OF CONTENTS DECLARATION .................................................................................................................... I ACKNOWLEDGEMENTS ................................................................................................... II TABLE OF CONTENTS ..................................................................................................... IV LIST OF FIGURES ............................................................................................................. XI LIST OF TABLES .......................................................................................................... XVIII ABBREVIATIONS .......................................................................................................... XIX ABSTRACT ...................................................................................................................... XXI PUBLICATIONS ........................................................................................................... XXIV CHAPTER 1 - INTRODUCTION ...................................................................................... 1 CHAPTER 2 - STREPTOMYCES AND THE OCCURRENCE OF GEOSMIN AND 2-METHYLISOBORNEOL IN AQUATIC ENVIRONMENTS .................................... 7 2.1 Introduction .................................................................................................................. 7 2.2 Actinobacteria and the genus Streptomyces: What are they? ....................................... 8 2.3 The ecological significance of actinobacteria ............................................................ 10 2.4 Geosmin and 2-MIB in drinking water supplies ........................................................ 11 2.4.1 Non-actinobacterial producers of geosmin and 2-MIB ....................................... 13 2.4.2 Geosmin and 2-MIB production by Streptomyces and other actinobacteria ....... 14 2.4.2.1 Conditions of production ............................................................................. 15 2.4.2.2 Pathways of biosynthesis ............................................................................. 18 2.4.2.3 Purpose of biosynthesis ................................................................................ 21 2.5 A terrestrial origin of Streptomyces and T&O compounds? ...................................... 22 2.6 Streptomyces and T&O production in aquatic environments ..................................... 23 2.6.1 The water mass .................................................................................................... 27 2.6.2 Vegetation ............................................................................................................ 28 2.6.3 Sediments and muds ............................................................................................ 29 IV 2.6.4 Other aquatic habitats .......................................................................................... 31 2.7 Conclusions ................................................................................................................ 33 CHAPTER 3 - EXPLORATORY MULTIVARIATE MODELLING OF PAST GEOSMIN AND 2-METHYLISOBORNEOL EPISODES IN GRAHAMSTOWN AND CHICHESTER DRINKING WATER RESERVOIRS ........................................ 35 3.1 Introduction ................................................................................................................ 35 3.2 Methods ...................................................................................................................... 39 3.2.1 Description of the drinking water reservoirs ....................................................... 39 3.2.2 Water quality data sets ......................................................................................... 41 3.2.3 Statistical analysis ................................................................................................ 43 3.3 Results ........................................................................................................................ 44 3.3.1 Overview of PLS models ..................................................................................... 44 3.3.2 PLS models for geosmin in Grahamstown Reservoir .......................................... 47 3.3.3 PLS models for 2-MIB in Grahamstown Reservoir ............................................ 53 3.3.4 PLS models for geosmin in Chichester Reservoir ............................................... 56 3.4 Discussion .................................................................................................................. 62 3.5 Conclusions ................................................................................................................ 69 CHAPTER 4 - STREPTOMYCES IN DRINKING WATER RESERVOIRS: DISTRIBUTION, ACTIVITY AND POTENTIAL CONTRIBUTION TO TASTE AND ODOUR EPISODES INVOLVING GEOSMIN AND 2-METHYLISOBORNEOL .............................................................................................. 71 4.1 Introduction ................................................................................................................ 71 4.2 Materials and methods ................................................................................................ 79 4.2.1 Sampling sites and collection .............................................................................. 79 4.2.2 Extraction of DNA from environmental samples ................................................ 84 4.2.3 Quantitative polymerase chain reaction (qPCR) protocol ................................... 87 V 4.2.4 Enumeration and isolation of Streptomyces......................................................... 91 4.2.5 Identification of Streptomyces-like isolates ......................................................... 96 4.2.5.1 Extraction of DNA from Streptomyces-like isolates