THE UNIVERSITY OF NEW SOUTH thesis/Dissertation Sheet Surname or Family name:LE Rrst ^ I Other narne/s: Abbreviation for degree as given in the University calendar: MSc | ScliooliBiotechnolo^^^^^^ and Biomolecular ScienoBS Faculty: Science Title:Community analysis and physiological characterisation of bacterial isolates from a nitrifying membrane bioreactor Abstract This thesis focuses on the identification of early colonisers on membrane surfaces used in wastewater treatment, as well as the physiological characterisation of bacterial cultures isolated from different micro- environments of a membrane bioreactor (MBR). The bacterial community composition of early biofilms on membrane surfaces under different hydrodynamic conditions (pressurised and non-pressurised) and of the activated sludge in an MBR were examined by culture-independent, molecular-based methods of PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and PGR cloning of 16S rRNA genes. A bench-scale, nitrifying MBR treating artificial waste was employed. The hollow fibre ultrafiltration membrane was made of polypropylene with an average pore diameter of 0.04 \im. Analysis of DGGE profiles of the sessile communities on membrane surfaces revealed that Tetrasphaera elongata species were important colonisers due to their ability to bind to membrane surfaces irrespective of the hydrodynamic context and exposure time. Interactions between isolates from the bioreactor and membrane surfaces were further investigated by characterising the physiological traits important in biofilm initiation and proliferation on membrane surfaces such as motility, auto-aggregation, co-aggregation, hydrophobicity and quorum sensing. Bacterial strains were isolated from floes and supernatant phases of the activated sludge as well as from pressurised membrane surfaces. Microbacterium sp. were prevalent in all culture collections. Physiological studies revealed Microbacterium sp. possesed high hydrophobicity and auto-aggregating activity that could contribute to their colonisation on membrane surfaces and persistence in floes. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968.1 retain all property 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 Abstracts International (this is applicable to doctoral theses only). .Ì2../..S..I..Z. QIQ. 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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 XILZIZ.OXQ. 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.' 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Signed Date UL^lZniQ Community analysis and physiological characterisation of bacterial isolates from a nitrifying membrane bioreactor Lan Hoang Le A thesis submitted in fulfilment of the requirements for the degree of Master of Science School of Biotechnology and Biomolecular Sciences Faculty of Science The University of New South Wales, Sydney, Australia April 2010 TABLE OF CONTENTS ACKNOWLEDGEMENT 3 LIST OF FIGURES 4 LIST OF TABLES 5 LIST OF ABBREVIATIONS 6 ABSTRACT: 7 CHAPTER 1 LITERATURE REVIEW 8 1.1 INTRODUCTION TO MEMBRANE BIOREACTORS (MBRS) 8 1.1.1 The MBR unit configuration 11 1.1.2 Advantages of membrane bioreactors: 12 1.1.3 Limitations of MBRs 14 1.2 MEMBRANE FOULING IN MBRS 15 1.2.1 Sludge characteristics 16 1.2.2 Membrane characteristics 16 1.2.3 Operating parameters 17 1.3 BIOFILM FORMATION IN MBR 17 1.3.1 Stages of bacterial biofilm formation 18 1.3.2 Biofilms on MBR membrane surfaces 19 1.4 THESIS OBJECTIVES 25 CHAPTER 2 EARLY COLONISATION OF MEMBRANE SURFACES 27 2.1 INTRODUCTION 27 2.2 MATERIALS AND METHODS 28 2.2.1 Membrane Bioreactor Set-up 28 2.2.2 Membrane sampling 29 2.2.3 Genomic DNA extraction 30 2.2.4 PGR for Denaturing Gradient Gel Electrophoresis (DGGE) 30 2.2.5 Denaturing Gradient Gel Electrophoresis (DGGE) 31 2.2.6 Gel extraction and sequencing of bands 31 2.2.7 Construction of a 16S rRNA gene clone library from sludge 32 2.2.8 Sequencing of DGGE bands and clone inserts 32 2.3 RESULTS 33 2.3.1 Bacterial community composition in sludge 33 2.3.2 Establishing a membrane sampling regime to examine colonisation 34 2.3.3 Sequencing of gel bands of interest (A, B and C) 39 2.3.4 Phylogenetic analysis of clone library of sludge 40 2.4 DISCUSSION 42 CHAPTER 3 ISOLATION AND CHARACTERISATION OF BACTERIA ISOLATED FROM MEBRANES, FLOCS AND SUPERNATANT OF AN MBR 45 3.1 INTRODUCTION 45 3.2 MATERIALS AND METHODS 47 3.2.1 Isolation of bacteria from membrane surfaces and activated sludge 47 3.2.2 Media and growth conditions 48 3.2.3 Physiological characteristics of isolates 48 3.2.4 Genomic DMA extraction 50 3.2.5 16S rRNA gene amplification 51 3.2.6 Sequencing ofl6S rDNA 51 3.2.7 Aggregation assays 52 3.3 RESULTS 53 3.3.1 Isolation and identification of cultivable bacteria 53 3.3.2 Detection ofN-acyl-L-homoserine lactone activity 55 3.3.3 Motility assays 56 3.3.4 Cell surface hydrophobicity 57 3.3.5 Auto-aggregation 58 3.3.6 Biofilm formation on polystyrene plates 59 3.4 DISCUSSION 61 CHAPTER 4 GENERAL DISCUSSION 64 REFERENCE 70 Acknowledgement I would like to thank Professor Staffan Kjelleberg for giving me the opportunity to join the Centre for Marine Bio-innovation four years ago. It has been a great discovery journey for me and I am very grateful for it. I would like to show my deep appreciation to my supervisors and co- supervisor, Dr. Diane McDougald and Dr. Mike Manefield for your huge support and patience. I am forever in debt to you for your guidance when I was most confused and had no confidence in myself. Thank you again and again and again. I would like to thank Professor Richard Stuetz and his phD student Minh Nhat Le from the School of Civil and Environmental Engineering for their help with the reactor. I would like to thank my family for their support for the past few years, to allow myself to pursuit my research passion. Thank you for having confidence in me. This is for you. Many thanks to friends and colleagues, especially people from CMB level 6: Nidhi, Leesia, Amy, Tran, Janice, Jerry, Krager. You are the ones that keep my experience in the lab so much enjoying: coffee, chocolate, tea, jokes. What else can one ask for at one's workplace? Last but not least, a hug to my boyfriend, thank you for your love, patience and confidence in me. List of Figures Figure 1.1 Flow diagram describing conventional wastewater treatment 9 Figure 1.2 MBR unit configuration 11 Figure 1.3 Development stages of a biofilm on a static substratum 19 Figure 1.4 Model of the central components of lux regulation in V. ficheri 24 Figure 2.1 Schematic diagram of the 26 litre, bench-scale MBR 29 Figure 2.2 Bacterial community fingerprints of sludge sample fi"om the MBR taken over a period of 85 days 33 Figure 2.3 Bacterial community profiles fi-om 4 day-old pressurised membrane 35 Figure 2.4 Bacterial community profiles from 4-hour