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Biofilms in water separation membrane processes from community structure and ecological characteristics to monitoring and control

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BIOFILMS IN WATER SEPARATION MEMBRANE PROCESSES: FROM COMMUNITY STRUCTURE AND ECOLOGICAL CHARACTERISTICS TO MONITORING AND CONTROL PANG CHEE MENG NATIONAL UNIVERSITY OF SINGAPORE 2007 BIOFILMS IN WATER SEPARATION MEMBRANE PROCESSES: FROM COMMUNITY STRUCTURE AND ECOLOGICAL CHARACTERISTICS TO MONITORING AND CONTROL PANG CHEE MENG (B. Eng (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CIVIL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements Acknowledgements I would like to express my heartfelt gratitude to my supervisor, Associate Professor Wen-Tso LIU for his intellectual guidance and invaluable advice in the course of this research project. Sincere thanks are extended to the members of the examination committee, as well as the two external examiners for reviewing this thesis. I would also like to thank the staff of the Environmental Engineering Laboratory in the Division of Environmental Science and Engineering, Ms Sally Toh, Mr. Chandrasegaran, Mr. Michael Tan, Ms Tan Xiaolan, Ms Tan Hwee Bee and Ms Lee Leng Leng, for their assistance in experimental support and with lab equipment. Analytical techniques imparted by Mr. Chen Chia-Lung and Ms Tan Fea Mein, as well as contributions by final year students, Cindy Koh Sim Yi, Hong Peiying, Guo Huiling and Ong Yun Qi are gratefully acknowledged. Finally, I wish to express my deepest thanks to my family, and my good friends in the lab for making this a memorable six years. i Table of Contents Table of Contents Acknowledgements . i Table of Contents . ii Summary ix List of Tables xv List of Figures xvi Abbreviations . xx Chapter 1: Introduction 1.1. Background .1 1.2. Problem Statements .5 1.2.1. Community structure of biofilms associated with membrane biofouling 1.2.2. Mechanisms for biofilm formation among dominant bacterial populations in membrane biofilms 1.2.3. Biofilm monitoring in membrane processes 1.2.4. Biofilm control in membrane processes 1.3. Research Objectives 10 1.4. Organization of Thesis 11 Chapter 2: Literature Review . 14 2.1. Microbial Biofilms 14 2.2. Biofilm Formation .14 2.2.1. Initiation of biofilm formation .14 ii Table of Contents 2.2.2. Cellular adhesion and biofilm formation .15 2.2.3. Bacterial motility and biofilm formation .17 2.3. Biofilm Heterogeneity .19 2.3.1. Addressing biofilm heterogeneity using non-destructive analytical approaches .22 2.4. Biofilms on reverse osmosis membranes .27 2.4.1. Biofouling on RO membranes .27 2.4.2. Microbial diversity in membrane biofilms 30 2.5. Antimicrobial resistance in biofilms .36 2.5.1. Diffusion limitation .36 2.5.2. Decreased growth rate .38 2.5.3. Expression of possible biofilm-specific resistance phenotypes .39 2.6. Biofilm Monitoring .41 2.6.1. Organic carbon-based biofilm monitoring .41 2.6.2. Surface-based biofilm monitoring .46 2.7. Biofilm Control .48 2.7.1. Conventional biofilm control measures .48 2.7.1.1. Chemical control strategies .48 2.7.1.2. Biological filtration: an organic carbon and nutrient limitation strategy .51 2.7.2. Alternative biofilm control measures 54 2.7.2.1. Quorum sensing and biofilm control 54 2.7.2.2. Titanium dioxide photocatalysis .57 2.8. Concluding Remarks .61 iii Table of Contents Chapter 3: Materials and Methods 62 3.1. Biofilm Samples 62 3.1.1. Membrane biofilms 62 3.1.2. Secondary effluent- and biofilter effluent-biofilms .65 3.2. Water Quality Analyses 65 3.3. PCR-Based Molecular Analyses .66 3.3.1. Sample collection and total community DNA extraction 66 3.3.2. Polymerase chain reaction .68 3.3.3. Terminal restriction fragment length polymorphism .69 3.3.4. 16S rRNA gene clone libraries and phylogeny analysis .70 3.4. Microscopy-Based Molecular Analyses .72 3.4.1. Fixation and embedding 72 3.4.2. Fluorescence in situ hybridization .72 3.4.3. Live/Dead staining .73 3.4.4. Lectin staining of biofilms .74 3.4.5. Microscopy and image analysis .74 3.5. Bacterial Isolation .75 3.6. Bacterial Strains and Growth Media .75 3.7. Pure Culture-Based Analyses 76 3.7.1. Motility assays .76 3.7.2. Cell surface hydrophobicity .77 3.7.3. Cell surface charge 78 3.7.4. Microtiter plate assay .78 3.7.5. BIOLOG GN2 MicroPlateTM assay .80 3.8. Biofilm Studies Using Continuous Flow Cell Systems 80 iv Table of Contents 3.9. Membrane Characterization 82 3.9.1. Polymer membranes 82 3.9.2. Atomic force microscopy 82 3.9.3. Contact angle measurements .83 3.9.4. Membrane surface zeta potential .83 3.10. Protein Analyses 84 3.10.1. Purification of AiiA protein .84 3.10.2. SDS-Polyacrylamide gel electrophoresis 85 3.10.3. Immobilization of AiiA protein onto glass substratum .86 3.11. Nucleotide Sequence Accession Numbers 87 Chapter 4: Community Structure Analysis of Reverse Osmosis Membrane Biofilms and the Significance of Rhizobiales Bacteria in Biofouling . 88 4.1. Abstract .88 4.2. Introduction .88 4.3. Results .90 4.3.1. Comparison of influent water quality 90 4.3.2. Biofilm community structure as revealed by 16S rRNA gene-based clone library and bacterial isolation 91 4.3.3. Biofilm community structure as revealed by 16S rRNA gene-based T-RFLP .93 4.3.4. Carbon substrate utilization patterns of biofilm isolates .96 4.3.5. Nitrogen reduction capability of biofilm isolates 99 4.4. Discussion .101 v Table of Contents Chapter 5: Biofilm Formation Characteristics of Bacterial Isolates Retrieved from a Reverse Osmosis Membrane 106 5.1. Abstract .106 5.2. Introduction .106 5.3. Results .108 5.3.1. Biofilm formation potential as determined by microtiter plates 108 5.3.2. Bacterial motility .111 5.3.3. Cell surface hydrophobicity and zeta potential 112 5.3.4. RO membrane properties .113 5.3.5. Comparison of RO2 and OUS82 biofilms on RO membranes 116 5.3.6. Fluorescently labeled lectin staining of RO2 biofilms 120 5.4 Discussion 122 5.4.1. Role of swimming motility in bacterial transport to RO membranes 122 5.4.2. Role of cell surface hydrophobicity and zeta potential in bacterial adhesion 123 5.4.3. Role of bacterial motility in biofilm formation .124 5.4.4. Biofilm studies in continuous flow cell systems .125 5.4.5. Implications for RO operation .127 Chapter 6: Biological Filtration Limits Carbon Availability and Affects Downstream Biofilm Formation and Community Structure 128 6.1. Abstract .128 6.2. Introduction .129 vi Table of Contents 6.3. Results .130 6.3.1. Performance of biofilter treating secondary effluent .130 6.3.2. Biofilm biomass as estimated by microtiter plate assay 132 6.3.3. Biofilm development dynamics and quantitative biofilm analyses .133 6.3.4. Biofilm community structure as revealed by FISH .138 6.3.5. Biofilm community structure as revealed by 16S rRNA gene clone libraries 140 6.3.6. Biofilm community structure as revealed by T-RFLP 142 6.4. Discussion .145 Chapter 7: Control of Pure Culture Biofilms using Enzymatic and Catalytic Antimicrobial Agents . 152 7.1. Abstract .152 7.2. Introduction .153 7.3. Results .155 7.3.1. Effect of AiiA enzyme on batch-cultivated biofilms .155 7.3.2. Effect of AiiA enzyme on biofilms cultivated under continuous flow conditions 160 7.3.3. Effect of TiO2 photocatalysis on batch-cultivated biofilms .163 7.3.4. Effect of TiO2 photocatalysis on biofilms cultivated in continuous flow cell systems .164 7.4. Discussion .167 vii Table of Contents Chapter 8: Conclusion and Recommendations . 172 8.1. Conclusions .172 8.1.1. Community structure analysis of reverse osmosis membrane biofilms and the significance of Rhizobiales bacteria in biofouling 172 8.1.2. 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Presented at Conference on Alternative and Conventional Anti-Fouling Strategies, Sep 13–15, 2004, Mülheim, Germany. 219 [...]... fundamental aspects of these membrane biofilms remains to be elucidated A more complete understanding into the diversity of biofilm microorganisms, together with their biofilm formation mechanisms and ecological selective advantages in this environment, is anticipated to aid in the development of more effective biofilm monitoring and biofouling control strategies in membrane processes 4 Introduction 1.2 Problem... Organization and UNESCO estimates, the world’s demand for water is growing three times as fast as the global population, and water shortages have become an increasingly serious problem in many parts of the world Indeed, one in six persons in the world did not have access to safe drinking water in 2001, and this number is anticipated to rise to four in ten persons by the year 2050 These are somber figures and. .. augmenting water sources by using membrane separation techniques have increased remarkably in response to the issue of water scarcity Membrane technology, especially reverse osmosis (RO), can in principle, produce high quality water that satisfies stringent regulatory and end-user standards Its major weakness is the accumulation of biological contaminants on the membrane surface leading to membrane. .. the need to search for alternative methods in the preparation of high quality drinking water In recent years, interests in augmenting water resources using non-traditional sources (such as brackish water, municipal wastewater and seawater) for either direct or indirect potable reuse have increased remarkably in response to the issue of water scarcity [1] This development is largely attributed to the... microcolonial structures in some parts of the biofilm even after 1 h of treatment Presently, the ubiquity and persistence of microbial biofilms have posed a unique challenge to membrane processes used in water purification and wastewater reclamation This thesis therefore hopes to develop a more vigorous understanding into the community structure associated with membrane biofilms, their metabolic characteristics, ... Summary Water is essential for human survival and vital to the sustainable development of human societies However, the demand for water far exceeds its current supply and this can be attributed to a number of factors, including an increasing global population, water pollution, poor water management practices and the slow transfer of technological expertise to needy countries In recent years, interests in. .. formation in the membrane environment and the associated microbial community structure are carefully addressed in this thesis As a first step, the biofilm community structure of several biofouled water purification membranes was characterized Among them, a lab-scale RO membrane treating wastewater effluent from a bioreactor was investigated using a polyphasic approach combining molecular techniques and bacterial... operational problem in RO membranes and is known to contribute to loss in membrane productivity through reduction in permeate fluxes, increase in differential pressure, decreased salt rejections, and membrane degradation [7] For systems operating with feed waters above 25oC, the fouling of membrane surfaces by biological contaminants, termed biofouling, becomes particularly important, as observed in those RO... facilities in the Mediterranean region [8] and Singapore [9] 2 Introduction Membrane biofouling occurs when microorganisms accumulate on the membrane surface and proliferate as biofilms The transition from planktonic cells to this sessile form of microbial life usually involves several stages including initial surface adhesion, microcolony formation, and the eventual maturation of microcolonies into an... submerged in the two wastewater streams were compared Using this method, biomass accumulated in carbon-limited BF biofilms was observed xi Summary to be consistently lower than SE biofilms Biofilms on glass slides were also collected from these two wastewaters in order to investigate the effect of organic carbon limitation on biofilm succession and community structure Based on T-RFLP fingerprinting, a . NATIONAL UNIVERSITY OF SINGAPORE 2007 BIOFILMS IN WATER SEPARATION MEMBRANE PROCESSES: FROM COMMUNITY STRUCTURE AND ECOLOGICAL CHARACTERISTICS TO MONITORING AND CONTROL PANG CHEE. BIOFILMS IN WATER SEPARATION MEMBRANE PROCESSES: FROM COMMUNITY STRUCTURE AND ECOLOGICAL CHARACTERISTICS TO MONITORING AND CONTROL PANG CHEE MENG. formation among dominant bacterial populations in membrane biofilms 6 1.2.3. Biofilm monitoring in membrane processes 8 1.2.4. Biofilm control in membrane processes 9 1.3. Research Objectives 10

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