Phylogenetic Diversity and Ecophysiology of Alphaproteobacterial Glycogen Accumulating Organisms in Enhanced Biological Phosphorus Removal Activated Sludge Systems Submitted by Simon Jon McIlroy Bachelor of Applied Science (Honours) La Trobe University A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy School of Molecular Sciences Faculty of Science Technology and Engineering La Trobe University, Bendigo, Victoria, 3552 Australia December 2010 Table of contents Abbreviations xiv Summary xvii Statement of authorship xix List of publications xx Acknowledgements xxiv 1.0 Introduction 1 1.1 The requirement for nutrient removal in the treatment of wastewater ......................... 1 1.2 The application of activated sludge to the treatment of wastewater............................. 2 1.2.1 Enhanced biological phosphorus removal ............................................................. 2 1.2.2 Nitrogen removal ................................................................................................... 6 1.3 The need for microbiological studies on P removal ..................................................... 7 1.4 Biochemical models of EBPR and PAO metabolism................................................... 7 1.4.1 Anaerobic metabolism of the PAO........................................................................ 8 1.4.1.1 The uptake of volatile fatty acids (VFAs)....................................................... 8 1.4.1.2 The source of anaerobic reducing equivalents................................................ 10 1.4.2 Aerobic metabolism of the PAO............................................................................ 11 1.4.3 The need for microbiological data in PAO modelling........................................... 11 1.5 How do we study the complex communities in EBPR systems?.................................. 12 1.5.1 Culture dependent methods.................................................................................... 12 1.5.2 Culture independent methods: The ‘Full rRNA cycle’ approach .......................... 14 1.5.2.1 Obtaining 16S rRNA gene sequence information from EBPR systems ......... 14 1.5.2.2 In situ analysis of populations......................................................................... 18 1.5.2.3 The functional phylogenetic unit: limitations of the 16S rRNA gene?........... 19 1.6 Which are the important PAO in EBPR systems? ........................................................ 20 1.6.1 The putative gammaproteobacterial PAO.............................................................. 20 1.6.1.1 The acinetobacterial PAO............................................................................... 20 1.6.1.2 Stentrophomonas sp. EBPR-1......................................................................... 23 1.6.2 The betaproteobacterial PAO................................................................................. 23 1.6.2.1 Lampropedia hyalina...................................................................................... 23 1.6.2.2 Candidatus ‘Accumulibacter phosphatis’....................................................... 28 1.6.2.2.1 Intraphylotypic diversity among ‘Accumulibacter’ ................................. 28 1.6.2.2.2. Anaerobic metabolism of ‘Accumulibacter’........................................... 30 1.6.2.2.3. Aerobic metabolism of ‘Accumulibacter’............................................... 32 1.6.2.2.4 Can ‘Accumulibacter’ denitrify?.............................................................. 33 1.6.2.3 Are members of the Dechloromonas PAO? ................................................... 35 i 1.6.3 Actinobacterial-related PAO.................................................................................. 36 1.6.3.1 Microlunatus phosphovorus............................................................................ 36 1.6.3.2 Tetrasphaera-related PAO.............................................................................. 37 1.6.3.2.1 Metabolism of the Tetrasphaera PAO..................................................... 38 1.6.4 Other possible PAO ............................................................................................... 39 1.7 The glycogen accumulating organisms (GAOs)........................................................... 40 1.7.1 The anaerobic metabolism of the GAO ................................................................. 41 1.7.2 Aerobic metabolism of the GAO ........................................................................... 42 1.8 The phylogenetic identity of the GAO.......................................................................... 42 1.8.1 The gammaproteobacterial GAO - Candidatus ‘Competibacter phosphatis’........ 43 1.8.1.1 Anaerobic metabolism of the ‘Competibacter’............................................... 45 1.8.1.2 Aerobic/anoxic metabolism of the ‘Competibacter’....................................... 45 1.8.2 Alphaproteobacterial-related GAO........................................................................ 48 1.8.2.1 Amaricoccus sp. .............................................................................................. 48 1.8.2.2 Sphingomonas-related GAO ........................................................................... 48 1.8.2.3 Defluviicoccus-related GAO........................................................................... 49 1.8.2.3.1 Anaerobic metabolism of the Defluviicoccus-related GAO .................... 50 1.8.2.3.2 Aerobic metabolism of the Defluviicoccus-related GAO ........................ 51 1.8.3 Other putative GAO............................................................................................... 52 1.9 Competition between the PAO and GAO populations in EBPR .................................. 53 1.9.1 Carbon source ........................................................................................................ 53 1.9.2 pH........................................................................................................................... 57 1.9.3 Temperature ........................................................................................................... 59 1.9.4 Carbon to phosphorus ratio (C:P)........... .............................................................. 60 1.9.5 Sludge retention time (SRT) .................................................................................. 60 1.9.6 Dissolved oxygen (DO) levels............................................................................... 61 1.10 Aims of this study ....................................................................................................... 62 2.0 Flow cytometry assisted identification of GAO in lab-scale EBPR systems.............. 63 2.1 Introduction................................................................................................................... 63 2.2 Materials and methods .................................................................................................. 65 2.2.1 Operation of the EPBR SBR reactors .................................................................... 65 2.2.2 Cell fixation ........................................................................................................... 66 ii 2.2.3 Fluorescence in situ hybridisation (FISH) ............................................................. 66 2.2.4 Fluorescence microscopy....................................................................................... 67 2.2.5 Quantitative FISH (qFISH).................................................................................... 67 2.2.6 Flow cytometry ...................................................................................................... 69 2.2.6.1 FISH labelling of cells .................................................................................... 69 2.2.6.2 Sorting of Alphaproteobacteria ...................................................................... 70 2.2.6.3 Sorting of ‘Competibacter’............................................................................. 70 2.2.7 Extraction of DNA from FACS sorted biomass .................................................... 70 2.2.8 16S rRNA gene clone library construction............................................................ 72 2.2.8.1 16S rRNA gene clone library PCR ................................................................. 72 2.2.8.2 Ligation and transformation............................................................................ 72 2.2.8.3 Plasmid extraction ......................................................................................... 74 2.2.8.4 Colony PCR .................................................................................................. 74 2.2.8.5 DNA electrophoresis ..................................................................................... 75 2.2.8.6 Sequencing and phylogenetic analysis .......................................................... 75 2.3 Results and discussion ................................................................................................ 76 2.3.1 SBR community compositions ............................................................................ 76 2.3.2 Flow cytometry sorting of the Alphaproteobacteria ........................................... 78 2.3.3 Clone library analysis of ALF968 FACS sorted cells ........................................... 78 2.3.4 Could FACS also assist in uncovering further diversity