Composition, dynamics and function of extracellular polymeric substances in drinking-water biofilms Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften – Dr. rer. nat. – vorgelegt von Witold Dariusz Michalowski geboren in Warschau Biofilm Centre – Aquatische Mikrobiologie der Universität Duisburg-Essen 2012 Die vorliegende Arbeit wurde im Zeitraum von Januar 2008 bis August 2012 im Arbeitskreis von Prof. Dr. Hans-Curt Flemming am Biofilm Centre der Universität Duisburg-Essen durchgeführt. Tag der Disputation: 06.11.2012 Gutachter: Prof. Dr. Hans-Curt Flemming Prof. Dr. Ulrich Szewzyk Vorsitzender: Prof. Dr. Jochen Stefan Gutmann “Water, taken in moderation, cannot hurt anybody” - Mark Twain - Acknowledgements ACKNOWLEDGEMENTS First of all I would like to express my gratefulness to Prof. Dr. Hans-Curt Flemming for setting up this interesting project and for his excellent supervision throughout my research. Hans- Curt, thank you for the inspiration, as well as the trust and the liberties to implement my own ideas, which I was able to present at the very reasonable number of conferences I’ve attended. Thank you to Jost Wingender for the numerous and inspiring discussions and support throughout all stages of my research. I am deeply indebted to both, Prof. Dr. Hans-Curt Flemming and Dr. Jost Wingender, for spending countless hours reviewing this manuscript and giving creative input. I wish to express my gratidude to Prof. Dr. Ulrich Szewzyk for accepting the task of being my co-referee. Many thanks to the Max Buchner Research Foundation for financial support. I would like to acknowledge Prof. Dr. Ursula Obst, Dr. Gerald Brenner-Weiß and Boris Kühl from the KIT for giving me the opportunity to identify proteins in their lab. Thanks to Daniel, Sascha and Mathias for their assistance during my first days in the lab, as well as to Miriam for lots of advice and encouragement especially towards the end of this work. I thank Barbara for the great work investigating parts of the dynamics of drinking- water biofilms and Dominik for the incorporation experiments with P. aeruginosa. Thank you to all current and former colleagues who worked with me in the department of Aquatic Microbiolgy and who provided a great atmosphere and lots of fun. All of you contributed a great deal to the success of this study. A special thanks to Silke for the support, the encouragement, the advice and the distraction whenever I needed it, as well as for proofreading this manuscript countless times. Silke, thank you for travelling the world with me! As last, I would like to thank my family and friends who had to suffer from my lack of time, especially during the more demanding periods of this work. i Table of contents TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................................................ i TABLE OF CONTENTS ....................................................................................................................................ii LIST OF TABLES .......................................................................................................................................... vi LIST OF FIGURES ....................................................................................................................................... viii GLOSSARY ................................................................................................................................................. xi ABSTRACT ............................................................................................................................................... xiii 1. INTRODUCTION ...................................................................................................................................... 1 1.1 The biofilm way of life ................................................................................................................... 1 1.2 Extracellular polymeric substances (EPS) ...................................................................................... 3 1.2.1 EPS components ..................................................................................................................... 6 1.2.2 EPS matrix formation ............................................................................................................ 14 1.2.3 Mechanical stability of biofilms ............................................................................................ 17 1.3 Biofilms in drinking water distribution systems .......................................................................... 19 1.4 Methods for biofilm and EPS analysis ......................................................................................... 22 1.4.1 Tools to study biofilm composition ...................................................................................... 23 1.4.2 Tools to study biofilm architecture ...................................................................................... 37 1.5 Aims of the study ......................................................................................................................... 41 2. MATERIALS ......................................................................................................................................... 43 2.1 Chemicals ..................................................................................................................................... 43 2.2 Cultivation media ........................................................................................................................ 46 2.3 Buffers and solutions ................................................................................................................... 47 2.4 Commercial kits ........................................................................................................................... 51 2.5 Enzymes ....................................................................................................................................... 51 2.6 Equipment ................................................................................................................................... 51 2.7 Software and databases .............................................................................................................. 54 3. METHODS ........................................................................................................................................... 55 ii Table of contents 3.1 Cultivation of drinking-water biofilms ......................................................................................... 55 3.2 Sampling of drinking-water biofilms ........................................................................................... 57 3.3 Determination of biofilm mass .................................................................................................... 57 3.4 Multi-element analysis of drinking water and biofilms............................................................... 58 3.5 Scanning electron microscopy of drinking-water biofilms .......................................................... 58 3.6 Confocal laser scanning microscopy (CLSM) ............................................................................... 58 3.7 Microbiological analysis .............................................................................................................. 59 3.7.1 Total cell count ..................................................................................................................... 59 3.7.2 Heterotrophic plate count .................................................................................................... 59 3.7.3 Culturability of P. aeruginosa pure cultures ......................................................................... 59 3.7.4 Culturability of P. aeruginosa in biofilms ............................................................................. 60 3.8 Isolation of extracellular polymeric substances (EPS) ................................................................. 60 3.9 Biochemical analysis of biofilms and EPS .................................................................................... 63 3.9.1 Proteins................................................................................................................................. 63 3.9.2 Carbohydrates ...................................................................................................................... 63 3.9.3 Uronic acids .......................................................................................................................... 64 3.9.4 DNA ....................................................................................................................................... 65 3.9.5 2-keto-3-deoxyoctonate (KDO) ............................................................................................ 66 3.10 Enzyme activity measurements ................................................................................................. 66 3.10.1 Glucose-6-phosphate dehydrogenase activity ................................................................... 66 3.10.2 Protease activity determination by zymogram gels ........................................................... 68 3.10.3 Fluorometric determination of enzyme activity ................................................................ 68 3.11 Molecular biology methods......................................................................................................