CORE Metadata, citation and similar papers at core.ac.uk Provided by Helsingin yliopiston digitaalinen arkisto Heterotrophic Bacteria Associated with Cyanobacteria in Recreational and Drinking Water Katri Berg Department of Applied Chemistry and Microbiology Division of Microbiology Faculty of Agriculture and Forestry University of Helsinki Academic Dissertation in Microbiology To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in Walter hall (2089) at Agnes Sjöbergin katu 2, EE building on December 11th at 12 o’clock noon. Helsinki 2009 Supervisors: Docent Jarkko Rapala National Supervisory Authority for Welfare and Health, Finland Docent Christina Lyra Department of Applied Chemistry and Microbiology University of Helsinki, Finland Academy Professor Kaarina Sivonen Department of Applied Chemistry and Microbiology University of Helsinki, Finland Reviewers: Professor Marja-Liisa Hänninen Department of Food and Environmental Hygiene University of Helsinki, Finland Docent Stefan Bertilsson Department of Ecology and Evolution Uppsala University, Sweden Opponent: Professor Agneta Andersson Department of Ecology and Environmental Sciences Umeå University, Sweden Printed Yliopistopaino, Helsinki, 2009 Layout Timo Päivärinta ISSN 1795-7079 ISBN 978-952-10-5892-9 (paperback) ISBN 978-952-10-5893-6 (PDF) e-mail katri.berg@helsinki.fi Front cover picture DAPI stained cells of Anabaena cyanobacterium and Paucibacter toxinivorans strain 2C20. CONTENTS LIST OF ORIGINAL PAPERS THE AUTHOR’S CONTRIBUTION ABBREVIATIONS ABSTRACT TIIVISTELMÄ (ABSTRACT IN FINNISH) 1 INTRODUCTION ..................................................................................................... 1 1.1 Cyanobacteria ........................................................................................................ 1 1.2 Cyanobacterial mass occurrences ......................................................................... 1 1.2.1 Bloom forming cyanobacteria ..................................................................... 1 1.3 Cyanobacterial toxins ............................................................................................ 2 1.3.1 Hepatotoxins ................................................................................................ 2 1.3.2 Neurotoxins ................................................................................................. 3 1.3.3 Other toxins of cyanobacteria ...................................................................... 4 1.3.4 Toxins analyses ............................................................................................ 5 1.3.5 Persistence of cyanobacterial toxins ............................................................ 6 1.4 Aquatic cyanobacteria and heterotrophic bacteria ................................................ 6 1.4.1 Aquatic bacteria ........................................................................................... 7 1.4.2 Interactions between heterotrophic bacteria and cyanobacteria .................. 7 1.5 Bacterial characterisation ...................................................................................... 8 1.5.1 Isolation of bacteria ..................................................................................... 9 1.5.2 Phenotypic analyses .................................................................................... 9 1.5.3 Molecular analyses in taxonomic identifi cation of bacteria ...................... 10 1.5.4 Genetic analyses in assessment of physiological features ......................... 11 1.5.5 Statistical analyses of multivariate data .................................................... 11 1.6 Problems associated with cyanobacterial water blooms ..................................... 12 1.6.1 Adverse human health effects associated with cyanobacterial water blooms ..................................................................... 12 1.6.2 Cyanobacterial water blooms and drinking water ..................................... 14 1.6.3 Cyanobacterial toxins and the drinking water treatment ........................... 14 1.6.4 Combined effects of cyanobacterial water blooms and heterotrophic bacteria on the drinking water quality ................................. 15 2 AIMS OF THE STUDY .......................................................................................... 18 3 MATERIALS AND METHODS ............................................................................ 19 3.1 Water samples and isolated bacterial strains ....................................................... 19 3.2 Methods ................................................................................................................ 19 4 RESULTS ................................................................................................................. 21 4.1 Heterotrophic bacteria associated with cyanobacteria ........................................ 21 4.1.1 Taxonomic assignment of the isolated bacterial strains ............................. 21 4.1.2 Bacteria degrading cyanobacterial hepatotoxins: Paucibacter toxinivorans gen. nov. sp. nov. .............................................. 21 4.1.3 Virulence potential of the heterotrophic bacteria associated with cyanobacteria ..................................................................................... 24 4.1.4 Effects of the heterotrophic bacteria on the growth of cyanobacteria ........ 25 4.2 Selectivity of the used growth media .................................................................. 26 4.3 Drinking water treatment effi ciency ................................................................... 28 4.3.1 Removal of toxins by the water treatment .................................................. 28 4.3.2 Removal of planktonic cells by the water treatment ................................... 29 4.3.3 Taxonomy of the heterotrophic bacteria isolated from drinking water ....... 30 5 DISCUSSION .......................................................................................................... 31 5.1 Characteristics of the isolated heterotrophic bacteria .......................................... 31 5.2 Drinking water treatment effi ciency .................................................................... 34 6 CONCLUSIONS ...................................................................................................... 36 7 ACKNOWLEDGEMENTS ..................................................................................... 38 8 REFERENCES ......................................................................................................... 39 APPENDIX 1 The bacterial strains analysed in this study, water type of their origin, isolation medium used, length of the acquired 16S rRNA gene sequence and its accession number, and the taxonomic assignment of the strains ..................... 52 LIST OF ORIGINAL PAPERS I K. A. Berg, C. Lyra, K. Sivonen, L. Paulin, S. Suomalainen, P. Tuomi, and J. Rapala. 2009. High diversity of cultivable heterotrophic bacteria in association with cyanobacterial water blooms. ISME J. 3: 314–325. II K. A. Berg, C. Lyra, R. M. Niemi, B. Heens, K. Hoppu, K. Erkomaa, K. Sivonen, and J. Rapala. Virulence genes in Aeromonas strains isolated from cyanobacterial water bloom samples associated with human health symptoms. Manuscript, submitted to ISME J. III J. Rapala, K. A. Berg, C. Lyra, R. M. Niemi, W. Manz, S. Suomalainen, L. Paulin, and K. Lahti. 2005. Paucibacter toxinivorans gen. nov., sp. nov., a bacterium that degrades cyclic cyanobacterial hepatotoxins microcystins and nodularin. Int. J. Syst. Evol. Microbiol. 55: 1563–1568. IV J. Rapala, M. Niemelä, K. A. Berg, L. Lepistö, and K. Lahti. 2006. Removal of cyanobacteria, cyanotoxins, heterotrophic bacteria and endotoxins at an operating surface water treatment plant. Wat. Sci. Technol. 54(3): 23–28. The original papers were reproduced with the kind permission of the copyright holders. THE AUTHOR’S CONTRIBUTION I Katri Berg participated in designing of the experiment, isolated the majority of the bacterial strains and performed their taxonomic identifi cation. She interpreted the results and wrote the paper. II Katri Berg participated in designing of the experiment and isolation of the bacterial strains. She also performed taxonomic identification of the bacteria and the statistical analyses of the data, interpreted the results and wrote the paper. III Katri Berg participated in designing of the experiment, studied the presence of fl agellae and bacteriochlorophyll of the strains, performed the toxin degradation tests and interpreted the results of the toxin analyses. She wrote a part of the paper. IV Katri Berg participated in the isolation of the bacterial strains, the toxin analysis and the taxonomic identifi cation of the strains. ABBREVIATIONS Adda (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10- phenyldeca-4,6-dienoic acid AH Aeromonas hydrophila BA Blood agar BLAST Basic local alignment search tool CAP Program for canonical analysis of principal coordinates CFU Colony forming unit DISTLMf Program for distance-based multivariate analysis for a linear model using forward selection ELISA Enzyme-linked immunosorbent assay EU Endotoxin unit GLC Gas-liquid chromatography HPLC High-performance liquid chromatography LD50 Lethal dose, 50% Microcystin-LR Microcystin with leusine as aminoacid 2 and arginine as amino acid 4 Microcystin-YR Microcystin with tyrosine as aminoacid 2 and arginine as amino acid 4