BIOFILM FORMATION AND CONTROL IN A MODEL DRINKING WATER DISTRIBUTION SYSTEM WITH PHOSPHORUS ADDITION FANG WEI NATIONAL UNIVERSITY OF SINGAPORE 2010 BIOFILM FORMATION AND CONTROL IN A MODEL DRINKING WATER DISTRIBUTION SYSTEM WITH PHOSPHORUS ADDITION FANG WEI (B.ENG) A THESIS SUBMITTED FOR THE DEGREE OF PHILOSOPHIAE DOCTOR DEPARTMENT OF CIVIL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENT I would like to express my gratitude and sincere appreciation to my supervisors, Associate Professor Hu Jiangyong and Professor Ong Say Leong for their outstanding guidance, invaluable encouragement, consistent understanding, caring and patience throughout my Ph.D study Many thanks go to all technicians, staff and students, especially Mr S.G Chandrasegaran, Ms Lee Leng Leng, Ms Tan Xiaolan at the Environmental Engineering Laboratory of Division of Environmental Science and Engineering, National University of Singapore, for their assistance and cooperation in the many ways that made this research study possible My deepest gratitude is also expressed to all my family members, especially my wife Shi Rui, who gave me endless love and support; and my parents, who gave me invaluable life and edification i TABLE OF CONTENTS Pages ACKNOWLEDGEMENT i TABLE OF CONTENTS ii SUMMARY vii NOMENCLATURE xi LIST OF FIGURES xii LIST OF TABLES xv LIST OF PLATES xvi CHAPTER ONE INTRODUCTION 1.1 Background 1.2 Objective and Scope of Study .5 1.3 Outline of Thesis CHAPTER TWO LITERATURE REVIEW .8 2.1 Overview of Biofilm Community 2.1.1 Biofilm Formation 2.1.2 Biofilm Compositions .12 2.1.3 Exopolysaccharides (EPS) and Biofilm Structure 14 2.2 Biofilm Formation in Drinking Water Distribution System (DWDS) 19 2.2.1 Development of Biofilm in DWDS 20 2.2.2 Biofilm-related Problems in DWDS 22 2.3 Biofilm Control in DWDS 23 ii 2.3.1 Mechanisms of Disinfection .25 2.3.2 Free Chlorine and Monochloramine Disinfections 27 2.3.3 Efficacies of Free Chlorine and Monochloramine Disinfections 28 2.3.4 Disinfection Resistance of Biofilm Cells 31 2.4 Effects of Nutrient Condition on Biofilm Formation in DWDS .33 2.4.1 Carbon-limiting and Phosphorus-limiting DWDS 34 2.4.2 Use of Orthophosphate as Corrosion Inhibitor 37 2.4.3 Potential Biological Effects of Addition of Phosphorus in DWDS 38 2.5 Current Status and Research Needs 43 CHAPTER THREE MATERIALS AND METHODS 49 3.1 Introduction 49 3.2 Experimental Setup .50 3.2.1 Annular Reactor System 50 3.2.2 Feed Water 52 3.2.3 Nutrient Stock 53 3.2.4 Free Chlorine Disinfection 53 3.2.5 Monochloramine Disinfection 53 3.3 Sampling and Analysis 54 3.3.1 Sampling Method 54 3.3.1.1 Water Sample 54 iii 3.3.1.2 Biofilm Sample .55 3.3.2 Water Sample Analysis .56 3.3.2.1 Heterotrophic Plate Count (HPC) 56 3.3.2.2 Free Chlorine 57 3.3.2.3 Monochloramine .57 3.3.2.4 pH and Temperature 57 3.3.2.5 Assimilable Organic Carbon (AOC) .57 3.3.2.6 Ion 58 3.3.3 Biofilm Sample Analysis 59 3.3.3.1 HPC 59 3.3.3.2 Total Carbohydrate Content (TCC) .59 3.3.3.3 Confocal Laser Scanning Microscopy (CLSM) .60 3.3.3.4 GN2 Microplate Community Level Assay .62 3.3.3.5 Fluorescence in Situ Hybridization (FISH) 66 3.3.3.6 Terminal Restriction Fragment Length Polymorphism (TRFLP) 68 3.3.4 Statistical Analysis 73 CHAPTER FOUR RESULTS AND DISCUSSIONS .74 4.1 Introduction 74 4.2 Effects of Phosphorus Addition on Microbial Growth .74 4.2.1 Biofilm and Planktonic Cell Growth 74 4.2.2 Biofilm EPS Quantity .80 iv 4.2.3 Biofilm Morphology and Structure 84 4.3 Effects of Phosphorus Addition on Disinfection Efficacy 90 4.3.1 Biofilm Development before Disinfections 90 4.3.2 Effects of Disinfection on Biofilm Cell Number 90 4.3.3 Effects of Disinfection on Biofilm EPS Quantity .96 4.3.4 Effects of Disinfection on Biofilm Morphology and Structure 101 4.3.5 Effects of Disinfection on Planktonic Growth 113 4.4 Effects of Phosphorus Addition on Biofilm Metabolic Potential 116 4.4.1 Substrate Utilization Pattern (SUP) 116 4.4.1.1 Phosphorus Addition 116 4.4.1.2 Free Chlorine Disinfection 119 4.4.1.3 Monochloramine Disinfection 123 4.4.2 Substrate Utilization Diversity 126 4.4.3 Metabolic Potential 128 4.4.4 Similarity of Metabolic Activity .129 4.5 Effects of Phosphorus Addition on Biofilm Community Structure 131 4.5.1 FISH 131 4.5.2 TRFLP .136 4.5.2.1 TRLFP Profiles .136 4.5.2.2 Phylogenetic Assignments 143 CHAPTER FIVE CONCLUSIONS AND RECOMMENDATIONS 147 v 5.1 Conclusions .147 5.2 Recommendations .149 REFFERENCES 151 PUBLICATIONS 180 vi SUMMARY Microbial regrowth is an important issue in drinking water distribution system (DWDS) management Biofilm formation on the internal surface of pipeline becomes a great concern as the majority of the microbial growth in DWDS is associated with biofilm development and biofilms are much more disinfectant resistant than their planktonic counterparts Biofilm formation in DWDS can be affected by various factors such as availability of nutrients, presence of disinfectants, pipeline materials, temperature and water flow rate, etc Phosphorus has been recently identified as another limiting nutrient other than organic carbon to microbial growth in DWDS As a commonly used corrosion inhibitor, phosphate is frequently introduced into DWDS and causes the increase of phosphorus concentration Phosphorus addition to DWDS has potential to increase the microbial growth and change the community structure However, the effects of phosphorus on biofilm formation in DWDS are still not well understood The purpose of this study is to provide an in-depth understanding of the biofilm formation and control in DWDS when phosphorus addition is implemented Annular reactors were used to simulate DWDS Phosphorus addition (3 µg l-1, 30 µg l-1 and 300 µg l-1 of phosphorus) was found to have a complicated effect on biofilm formation (especially for 30 µg l-1 and 300 µg l-1 of phosphorus vii additions) Phosphorus addition can promote the biofilm cell growth (cell count increased by about log with addition of 30 µg l-1 and 300 µg l-1 of phosphorus.) However, the addition of 30 µg l-1 and 300 µg l-1 of phosphorus caused decrease in exopolysaccharides (EPS) quantity by 81% and 77%, respectively The results of biofilm structure analysis showed that the addition of 30 µg l-1 and 300 µg l-1 of phosphorus induced thicker and less homogeneous biofilms with more biomass The addition of µg l-1 of phosphorus, on the other hand, was found to have minor effects on the above parameters examined The results in this study confirmed that the addition of phosphorus to DWDS has a potential to increase the bacterial cell number and deteriorate the drinking water quality In the biofilm control study, free chlorine and monochloramine were used as disinfectants The disinfection efficacies of both free chlorine and monochloramine were increased when phosphorus was added into the reactor systems At the same disinfectant dosages, monochloramine showed greater biofilm removal efficiency than free chlorine (0.86 and 1.32 log cell number reduction for 0.5 mg 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Use of Live/Dead staining for quantifying biofilm bacterial viability Advances in Asian Environmental Engineering, 5(1), 25-30 Fang, W., Hu, J.Y and Ong, S.L (2007) Effects of environmental stress conditions on Pseudomonas Aeruginosa biofilm Advances in Asian Environmental Engineering, 6(1), 25-30 Hu, J.Y., Fang, W and Yu, B (2008) Biofilm control using chlorine-based disinfectants in model drinking water distribution systems Water Science and Technology: Water Supply, 8(5), 489-497 Fang, W., Hu, J.Y and Ong, S.L (2009) Influence of phosphorus on biofilm formation in model drinking water distribution systems Journal of Applied Microbiology, 106(4), 1328-1335 Fang, W., Hu, J.Y and Ong, S.L (2010) Effects of phosphorus on biofilm disinfections in model drinking water distribution systems Journal of Water and Health, 8(3), 446-454 Hu, J.Y., Fang, W and Ong, S.L (2011) Evaluation of metabolic activity and 180 Publications community structure of drinking water biofilm with the addition of phosphorus and disinfection treatments In preparation 181 ... formation of microbial aggregates, attachment to surfaces, structural stability and spatial arrangement of biofilm structure and serve as a protective barrier against desiccation and retards access... Biofilm formation begins with the attachment of free-floating bacterial cells to a surface And this attachment is followed by growth into a mature, structurally complex biofilm and culminates in the... of phosphorus to DWDS has a potential to increase the bacterial cell number and deteriorate the drinking water quality In the biofilm control study, free chlorine and monochloramine were used as