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Investigation of Initial Attachment and Biofilm Formation of Mesophilic Leaching Bacteria in Pure and Mixed Cultures and Their Efficiency of Pyrite Dissolution

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Investigation of Initial Attachment and Biofilm Formation of Mesophilic Leaching Bacteria in Pure and Mixed Cultures and Their Efficiency of Pyrite Dissolution Attachment of acidophilic bacteria to solid substrata Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften - Dr. rer. Nat. - vorgelegt von Nanni Noël geboren in Oberhausen Fakultät für Chemie der Universität Duisburg-Essen 2013 Die vorliegende Arbeit wurde im Zeitraum von Oktober 2008 bis September 2013 im Arbeitskreis von Prof. Dr. W. Sand Fakultät für Chemie, Biofilm Center der Universität Duisburg-Essen durchgeführt. Tag der Disputation: 18.12.2013 Gutachter: Prof. Dr. W. Sand Prof. Dr. A. Schippers Vorsitzender: Prof. Dr. S. Barcikowski … to my mum, Ben & in memory of my dad … -Science is the poetry of reality- (Richard Dawkins) I Acknowledgements Acknowledgments At first, I would like to thank my Ph.D. supervisor Prof. Dr. Wolfgang Sand not only for the guidance and support during this study, but also for the understanding of private problems in hard times of my life. Thanks for the warm words and the free space which I needed to find my own way to deal with the situation. I’ am grateful to Prof. Dr. Axel Schippers for accepting the task for being my co-adviser. Special thanks go to Dr. Mario Vera for his great support during this thesis and the helpful discussions about the communication pathways of bacteria. Thanks for proofreading and discussions of this work. I ‘am grateful that I was allowed to travel to Chile and to be received in a very friendly way in a foreign country. I’ am grateful to Fraunhofer-Umsicht, Oberhausen for financial support. I would like to thank Dr. Holger Wack, Damian Hintemann and Frank Pape. It was a pleasure to work with them in different biocorrosion projects during the last years. Thanks to Prof. Dr. N. Guiliani for a nice cooperation via DAAD and helpful discussions about communication pathways of bacteria. Great thanks to Prof. Dr. Wolfgang Streit, Universität Hamburg, Claudia Hornung and Frederike Haak for the help with the biosensor bacterium HH01. Thanks to Prof. Dr. Thorsten Schmidt, Jens Laaks and Xochitli Osorio for the help with the GC-MS and the interpretation of MS- spectra. I would like to thank all the persons from Prof. Sand’s working group who helped and supported me during the last years whether they are still members of this group (Petra, Qian, RuiYong, Sören, Til) or already find their ways in their working career (Bianca, Claudia, David, Igor, Jürgen, Mariël & Felipe). Special thanks go to: Bianca, who was every time interested in my work, pushed me forward and for her talent to be a “headshrinker”. Thanks for this huge friendship and also for proofreading of this thesis. Bea, “my little thunderstorm” for her big heart, helpfulness and friendship. Claudia, for discussions about “the great At. caldus”, molecular biology or the unpredictability’s of life. Andrzej and Christian, for technical advice and handling with the AFM-EFM, IC or HPLC. Also thanks for funny discussions about diverse passions, music/movies and drinks. That made long days during sample preparations/processing much easier. Igor, Jens, Mark and Robert for funny good times especially during sampling sessions or after work to keep the “bad twins” alive. Conny & Jürgen and Mariël & Felipe, for a great friendship and moral support during the last years. Mia, “my travel companion”, for the right interruptions of work, for travelling around the world to enjoy the most beautiful things of life. All my friends, above all “J”, Farina, Karin, Sandi, for their never ending support during the last years. The biggest thanks go to my family, first of all: my lovely mum, my brother Ben and his wife Maike, my granny and my dad -it was not granted to him to undergo this day- II Table of Contents Table of Contents Acknowledgments .............................................................................................................. II Table of Contents ................................................................................................................... III List of Figures ........................................................................................................................ VI List of Tables ...................................................................................................................... VIII Glossary ............................................................................................................................ IX Abstract ............................................................................................................................. XI 1. Introduction ....................................................................................................................... - 1 - 1.1. Bioleaching .................................................................................................................. - 1 - 1.2. Bioleaching Mechanisms ............................................................................................. - 1 - 1.3 Attachment and biofilm formation on pyrite ................................................................ - 5 - 1.4 Quorum sensing (QS) ................................................................................................... - 7 - 1.4.1 N-acyl homoserine lactones (AHLs) and the LuxI/R- QS-system ....................... - 7 - 1.4.2 A novel QS system regulated by α-hydroxyketone signaling ............................... - 8 - 1.4.3 Quorum sensing and bioleaching .......................................................................... - 9 - 1.5 Bioleaching community ................................................................................................ - 9 - Generals .............................................................................................................................. - 9 - Diversity of leaching organisms .................................................................................. - 11 - Moderately thermophilic leaching organisms .............................................................. - 11 - 2. Aims of this study ........................................................................................................... - 13 - 3. Materials & Methods ....................................................................................................... - 14 - 3.1. Microorganisms and growth ...................................................................................... - 14 - Purity control .................................................................................................................... - 15 - Cell harvest ....................................................................................................................... - 15 - 3.2 Substrate Pyrite ........................................................................................................... - 15 - 3.3 Part 1: Attachment and Leaching experiments ........................................................... - 19 - 3.3.1 Attachment experiments ..................................................................................... - 19 - III Table of Contents Attachment to pyrite grains ..................................................................................... - 19 - Attachment to pyrite coupons .................................................................................. - 19 - 3.3.2 Leaching experiments ......................................................................................... - 19 - 3.3.3 Precolonization experiments ............................................................................... - 20 - 3.3.4 Analytical procedures ......................................................................................... - 23 - Total cell count determination (TTC) ...................................................................... - 23 - pH determination ..................................................................................................... - 23 - Iron ion determination ............................................................................................. - 23 - Sulfate and thiosulfate determination ...................................................................... - 23 - 3.4 Visualization techniques ........................................................................................ - 24 - 3.4.1 4’, 6-diamino-2-phenylindole (DAPI)- staining ............................................. - 24 - 3.4.2 Fluorescent-in-Situ-Hybridization FISH ........................................................ - 24 - 3.4.3 Lectin staining ................................................................................................ - 26 - 3.4.4 Biofilm formation on “Floating-filters” ......................................................... - 26 - 3.4.5 Epifluorescence microscopy (EFM) ............................................................... - 27 - 3.4.6 Confocal-Laser-Scanning-Microscopy (CLSM) ............................................ - 27 - 3.5 PartII: Effect of N-acetyl-homoserine-lactones (AHLs) and other signal molecules .... - 27 - 3.5.1 Effect of AHLs ............................................................................................... - 27 - Leaching experiments .............................................................................................. - 28 - Attachment experiments .......................................................................................... - 28 - Floating filter experiments ...................................................................................... - 28 - 3.5.2. Extraction of AHLs ......................................................................................
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