A Dissertation entitled Role of Extracellular Polymeric Substances (EPS) on Biofilm Disinfection in a Model Drinking Water Distribution System by Zheng Xue Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Civil Engineering _________________________________________ Dr. Youngwoo Seo, Committee Chair _________________________________________ Dr. Ashok Kumar, Committee Member _________________________________________ Dr. Cyndee L. Gruden, Committee Member _________________________________________ Dr. Dong-Shik Kim, Committee Member _________________________________________ Dr. Jeffrey G. Szabo, Committee Member _________________________________________ Dr. Patricia Komuniecki, Dean College of Graduate Studies The University of Toledo December 2012 Copyright 2012, Zheng Xue This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Role of Extracellular Polymeric Substances (EPS) on Biofilm Disinfection in a Model Drinking Water Distribution System by Zheng Xue Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Civil Engineering The University of Toledo December 2012 Biofilms are undesirable but ubiquitous in drinking water systems. This study investigated the role of extracellular polymeric substances (EPS) in the biofilm life cycle, including planktonic cells, attached biofilm, detached biofilm clusters and redistributed biofilm, in a model distribution system with minimal disinfectant residuals. EPS contributed to bacterial surface properties, biofilm structural characteristics, disinfectant diffusion and reaction, organic matter retention and utilization, hence playing pivotal roles in bacterial resistance to disinfectants. Strains from an opportunistic pathogen, Pseudomonas aeruginosa with different EPS secretion capabilities were tested. Two major components in P. aeruginosa EPS, polysaccharides and proteins, both reacted rapidly with chlorine while monochloramine reacted specifically with proteins. The impact of biofilm EPS reactivity with disinfectants on disinfection efficacy was evaluated by monitoring planktonic bacteria viability, disinfectant decay, biofilm viability, biofilm structure, and detached biofilm viability as well as their redistribution, systematically iii during the disinfection process. The obtained results suggested that the presence of EPS increased the resistance of planktonic bacteria, biofilm and detached biofilm to both chlorine and monochloramine. The EPS reactivity led to different protection approaches for bacterial cells, acting either as a disinfectant consumer (for chlorine inactivation) or limiting access to reactive sites on a cell membrane (for monochloramine inactivation). The biofilm structure characterization using confocal laser scanning microscopy (CLSM) revealed that EPS production affected biofilm structure, specifically surface roughness, surface area to volume ratio and average diffusion distance. These structural characteristics were closely related to overall biofilm viability and the spatial distribution of viability within biofilms. Although the overall viable ratios were similar under the two disinfectants for each strain, monochloramine penetrated deeper into biofilm matrix than chlorine regardless the quantity of EPS content, showing a higher inactivation efficacy in the middle section of biofilms. However, chlorine was more efficient in controlling planktonic and detached cluster viability than monochloramine. The combined results suggested that different reactivity of biofilm EPS with disinfectants influenced the susceptibility of both biofilm and detached biofilm during the disinfection practices. This study provides valuable insight for both fundamental studies of biofilm life cycle and disinfection practices to optimize water quality maintenance in distribution systems. iv This dissertation is dedicated to my parents for their endless love, support and encouragement since the very beginning of my studies. v Acknowledgements I would like to thank all of the people who helped make this dissertation possible. First, I wish to thank my advisor, Dr. Youngwoo Seo for all his guidance, encouragement, support, and patience. His sincere interests in research and education have been a great inspiration to me. Also, I would like to thank my committee members, Dr. Ashok Kumar, Dr. Cyndee Gruden, Dr. Dong-Shik Kim, and Dr. Jeffrey Szabo, for their very helpful insights, comments and suggestions. Additionally, I would like to acknowledge all of those people who assisted, advised, and supported my research over the years: Dr. Andrea Kalinoski, for training and assisting me with confocal laser scanning microscopy; Dr. Mau-yi Wu, for valuable advice and suggestions; Dr. Yakov Lapitsky, for guidance on the Zetasizer analysis; Tammy Phares, for providing very helpful support in conducting experiments; and Sean Linkes, for technical support at the Flow Cytometry Core Facility. Finally, I would like to thank my colleagues and friends, Christopher Hessler, Varunraj Sendamangalam, Kimberly Coburn, Bin Li and Rui Zheng, who all provided invaluable assistance, support and suggestions throughout this process. Finally, I would like to thank my parents, Zaiquan and Ruiwei, for their support and encouragement. I could not have completed this effort without their love, tolerance, and enthusiasm. vi Contents Abstract ............................................................................................................................. iii Acknowledgements ............................................................................................................ vi Contents ............................................................................................................................ vii List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiii List of Abbreviations ....................................................................................................... xvi List of Symbols .............................................................................................................. xviii Preface ............................................................................................................................. xix 1 Introduction ...................................................................................................................... 1 2 Objective and Significance .............................................................................................. 7 3 Pseudomonas aeruginosa Inactivation Mechanism is Affected by Capsular Extracellular Polymeric Substance Reactivity with Chlorine and Monochloramine ....... 12 Abstract ......................................................................................................................... 12 3.1 Introduction ............................................................................................................. 13 3.2 Materials and Methods ............................................................................................ 15 3.2.1 Bacterial Culture ............................................................................................... 15 3.2.2 Preparation of Disinfectant Solutions ............................................................... 16 3.2.3 Batch Disinfection Experiments ....................................................................... 16 vii 3.2.4 Modeling with GInaFiT .................................................................................... 17 3.2.5 Bacteria Cell Staining ....................................................................................... 18 3.2.6 EPS Extraction and Characterization ............................................................... 19 3.2.7 Disinfectant Decay by Extracted EPS .............................................................. 20 3.2.8 FTIR Spectroscopy ........................................................................................... 20 3.2.9 Statistical Analysis ........................................................................................... 21 3.3 Results ..................................................................................................................... 21 3.3.1 Culture and EPS Properties .............................................................................. 21 3.3.2 Inactivation Kinetics ......................................................................................... 22 3.3.3 Model Fitting .................................................................................................... 25 3.3.4 Viability Analysis by Fluorescent Staining ...................................................... 27 3.3.5 Disinfectant Decay ........................................................................................... 27 3.3.6 FTIR Analysis................................................................................................... 30 3.4 Discussion ............................................................................................................... 35 3.5 Conclusion ..............................................................................................................