Analysis of Biofilm Communities in Breweries Markus Timke Osnabrück November 2004 Analysis of Biofilm Communities in Breweries Dissertation Zur Erlangung des akademischen Grades eines Doctor rerum naturalium (Dr. rer. nat.) eingereicht am Fachbereich Biologie/Chemie der Universität Osnabrück von Markus Timke Osnabrück, November 2004 Dissertation vorgelegt dem Fachbereich Biologie/Chemie der Universität Osnabrück Mündliche Prüfung: 21. Dezember 2004 Promotionskommission: Prof. Dr. K. Altendorf, Prof. Dr. H.-C. Flemming, Priv.-Doz. Dr. A. Lipski, Prof. Dr. F.-J. Methner Für meine Familie Preface Preface This work was part of the joint research project “Development of innovative strategies for an efficient and environmentally sound abatement of biofilms in the food industry considering the filling of beer exemplarily” (title in German: “Entwicklung innovativer Strategien zur effizienten und umweltschonenden Bekämpfung von Biofilmen in der Lebensmittelindustrie am Beispiel der Bierabfüllung”). The project partners were the Lehrstuhl Aquatische Mikrobiologie, Universität Duisburg-Essen, Privatbrauerei A. Rolinck GmbH & Co, Steinfurt, the Bitburger Brauerei Th. Simon GmbH, Bitburg and the Department of Microbiology, University of Osnabrück. The results are presented in nine Chapters. The Chapters 1 to 4 are manuscripts and represent the main part of this study. Additional data are reported in Chapters 5 to 9 to complete the picture of microbial communities of brewery biofilms obtained in this study. Acknowledgments First of all, I want to express my sincere thanks to many people, all of which contributed to this work in a scientific or private way. I thank Prof. Dr. Karlheinz Altendorf for providing the facilities for this study. He gave me the chance to come to Osnabrück and to start my Ph.D. studies in his department. In addition, he enabled me to participate in conferences. I am grateful to Priv.-Doz. Dr. André Lipski for introducing me to the fascinating world of microbial ecology, bacterial taxonomy and lipid biomarkers. He was always willing to discuss problems. Furthermore, he showed me ways to present and to compress data. I want to acknowledge Ngoc Quynh Wang-Lieu in a warm and special way. This thesis would not have reached its volume without her help. I want to thank all members of the joint-research project for the interesting and inspiring meetings we had: Prof. Dr. Karlheinz Altendorf, Prof. Dr. Hans-Curt Flemming, Mr. Heribert Frank, Priv.-Doz. Dr. André Lipski, Prof. Dr. Frank-Jürgen Methner, Mr. Karl Mock, Dr. Adriana Tamachkiarow, Mr. Gerhard Schröder and Mr. Dietmar Sommer. It was very nice to have so many people being interested in new data. In addition, they enabled me to get insights in the brewing industry and in business aspects. In this context, I want to acknowledge the "Deutsche Bundesstiftung Umwelt” for the financially support of this joint-research project. I Preface I am grateful to Mrs. Maria Thünemann for answering all my questions concerning the bottling plant and for providing some samples. I thank Prof. Dr. Clay Fuqua for sending me the AHL test system and Dr. Alfons Ahrens, Prof. Dr. Frank-Jürgen Methner, Dr. Johannes Hinrichs, Mr. Karl Mock and Prof. Dr. Ulf Stahl for kindly providing me some reference strains. I appreciate Dr. Udo Friedrich for many helpful suggestions and his advice. I want to thank all members of the Department of Microbiology for their constant cooperativeness and the worm working atmosphere. Special thanks goes to my lab colleagues during the years: Michèle Friedrich, Udo Friedrich, Ngoc Quynh Wang-Lieu, Dorothee Wolking, Claudia Knief, Julia Piehl, César Rodriguez-Sánchez, Ilka Winkler, Annegret Wachlin, Ramon Klein, Peter Herzog and Alexandra Kötter. Sincere thanks are given to my friends. I can just mention some aspects of their support and significance to me, otherwise it would go beyond the scope of this acknowledgments: Dr. Jürgen Clausen, who shares my interest in numismatics; Michèle and Udo Friedrich, they taught me the meaning of “foppen”; Marc Vor der Brüggen, an expert of electronic data processing and an inexhaustible source of news around the university; Thomas Krebstakies and Dr. Marc Bramkamp, for nice evenings in taverns of Osnabrück; Manuela Vogt, Gjon Blakqori, Kai Lund-Jensen, Marcel Wittke, Dr. Annika Wülfing and César Rodriguez-Sánchez, for their friendship over the years. I appreciate Ulrike and Axel Oschmann for their friendship, nice weekends, “survival holidays“ and for the certainty to have a home in Bremen. In special, I want to express my deepest thanks to my family and Dianes family, which has become mine, too. They always supported me over the years. Finally, I want to thank my dearest lady and wife Diane Timke. I will always be grateful for her love, undivided support and understanding. Osnabrück, November 2004 Markus Timke II Table of contents Table of Contents Summary ............................................................................................................................... V Zusammenfassung ..............................................................................................................VII General Introduction.............................................................................................................IX Chapter 1...............................................................................................................................1 Microbial Composition of Biofilms in a Brewery Investigated by Fatty Acid Analysis, Fluorescence in Situ Hybridization and Isolation Techniques Chapter 2.............................................................................................................................19 Community Structure and Diversity of Biofilms from a Beer Bottling Plant Revealed by 16S rRNA Gene Clone Libraries Chapter 3.............................................................................................................................43 Fatty Acid Analysis and Spoilage Potential of Mature Biofilms from Two Breweries Chapter 4.............................................................................................................................67 Characterization of Yeasts from Beer Bottling Plant Associated Biofilms Chapter 5.............................................................................................................................81 Isolation and Identification of Bacterial Isolates from Two Breweries Chapter 6.............................................................................................................................89 Detection of Acyl-Homoserine-Lactones Secreting Isolates Chapter 7.............................................................................................................................97 Isolation of Enterobacteriaceae from a Beer Bottling Plant and Their Capability to Grow in Beer Chapter 8...........................................................................................................................107 Occurrence and Quantitative Importance of Acetic Acid Bacteria in Brewery Biofilms Chapter 9...........................................................................................................................115 Fluorescence in Situ Detection and Isolation of Anaerobic Beer-Spoiling Bacteria Appendix ............................................................................................................................ XV III IV Summary Summary The main objective of this study was the characterization of surface associated microbial communities in breweries. In addition, the beer-spoiling potential of isolated strains and biofilm samples was investigated. Some studies reported the identity of cultivatable organisms from industrial plants. However, there were no data available about the composition of biofilm communities from these habitats for cultivation-independent techniques. Consequently, the fatty acid methyl esters (FAMEs) analysis, the fluorescence in situ hybridization (FISH) and the construction and investigation of 16S rRNA gene clone libraries were applied to reveal the structure of these communities. All of these methods have different advantages and therefore, they complement each other to get a more reliable picture of the biofilm communities. The cultivation method was included in this study because it enables a verification of results from other studies. Furthermore, the obtained strains are genuine brewery isolates and can be used for physiological tests. Isolates were obtained from seven different sample sites (Chapter 1 and 5). They were identified and affiliated to 25 different genera. Some of these strains were inoculated in beer but none of them was able to grow in it (Chapter 1 and 5). However, these strains can still be harmful for the industry, e.g. if they are able to form biofilms. This aspect was investigated by analyzing the potential of the isolates to produce acyl-homoserine lactones (AHLs) (Chapter 6). These quorum sensing mediating molecules are involved in the maturation process of biofilms. Indeed, some strains were found to secrete these autoinducer molecules, they mainly belonged to the genus Pseudomonas. An abundant proportion among the isolates was constituted by members of the Enterobacteriaceae (Chapter 7). In the beginning of this study, there was a minor suspicion concerning their beer-spoiling potential. Indeed, all isolated Enterobacteriaceae were found to be able to multiply in non-alcoholic beer under access of oxygen but they represented