Uptake, physiological responses and technical application of the aquatic fungus Mucor hiemalis EH5 for the removal of cyanobacterial toxins vorgelegt von MSc Evelyn Balsano geb. in Bozen, Italien von der Fakultät III – Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktor der Naturwissenschaften - Dr. rer. nat. - genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr. Juri Rappsilber Gutachter: Prof. Dr. Stephan Pflugmacher Lima Gutachter: Prof. Dr. Enamul Hoque Gutachterin: Prof. Dr.–Ing. Vera Meyer Tag der wissenschaftlichen Aussprache: 05. Oktober 2017 Berlin 2017 Auch eine Reise von tausend Meilen beginnt mit dem ersten Schritt ACKNOWLEDGEMENTS This study was conducted at the Department of Ecological Impact Research and Ecotoxicology and was funded by the Elsa-Neumann-Scholarship (formally known as Nachwuchsförderungsgesetz-NaFöG) and in part by the Studienbeihilfen für postuniversitäre Ausbildungen, Autonome Provinz Bozen Südtirol. I especially thank my academic supervisor Prof. Dr. Stephan Pflugmacher Lima for the opportunity to work in his well-equipped department on such a fascinating and innovative topic. I am thankful for the guidance and advice during the years and his confidence in my work. I further very much appreciated the possibility to present the Green Liver Systems on the exhibition IFAT in Munich, the inauguration of Felidae in Barnim and other occasions at the TU Berlin, thereby offering me the opportunity of being also involved in other fruitful outcomes of our research group. I am deeply grateful to my co-supervisor Prof. Dr. Enamul Hoque for the constant support and interest in the progress of my thesis throughout all these years. I am especially thankful for always welcoming me at the Helmholtz Zentrum München as well as introducing me to his wonderful family. Special thanks go to Dr. Maranda Esterhuizen-Londt for being my direct contact, her patient guidance, constant availability and amazing support throughout the course of this study. I also want to express my gratitude for all the invaluable corrections and suggestions on the manuscripts and this thesis. I further express my sincere gratitude to Dr. Valeska Contardo-Jara for her fruitful inputs and constructive discussions. Our laboratory technicians merit special recognition for always being ready to help and assist with any technical and supportive needs, Sandra Kühn especially for the help with the LC-MS/MS measurements and Mahboobeh Behmaneshfard for her unique kindness. I I further thank… All the staff members, especially Anja, Annette, Azam, Beheshteh, Cecilia, Cielo, Maranda, Mahboobeh, Mi-Hee, Sandra, Suhana, Suseth, and Valeska for their companionship, the pleasant work atmosphere, inspiring and fun lunches, and very good team spirit throughout the course of my entire thesis. ! My former lab and office colleague from the University of Vienna, Stephan Mokesch for his motivating scientific talks, his honest interest in the development of my work and also for reviewing the introduction part of this thesis. My friends for the irreplaceable encouragement, emotional support, and beneficial distractions from work when I needed it the most, especially Anna, Carlo, Dani, Esi, Kurt, Magda, Masha, Nora, Stephan, and Suhana. Last but most important, I am happy and grateful of having met Daniel at the peak of my PhD journey, whom I have always been able to count on through thick and thin ever since. And of course, I particularly thank my FAMILY for always being here and supporting me unconditionally in all the steps I take. II LIST OF MANUSCRIPTS This doctoral thesis is presented in cumulative form and is based on the following three manuscripts, referred to in the text by their corresponding Roman numerals. The three manuscripts, which form separate chapters in the thesis, have been published in international peer-reviewed journals and are inserted herein as their postprint version. I) Toxin resistance in aquatic fungi poses environmentally friendly remediation possibilities: A study on the growth responses and biosorption potential of Mucor hiemalis EH5 against cyanobacterial toxins. Evelyn Balsano, Maranda Esterhuizen-Londt, Enamul Hoque, and Stephan Pflugmacher International Journal of Water and Wastewater Treatment, 2015, 1(1), 1-9 Doi: 10.16966/2381-5299.101 This work is licensed under the Creative Commons Attribution 4.0 International License. CC BY 4.0 II) Fungal pellets as potential tools to control water pollution: Strategic approach for the pelletization and subsequent microcystin-LR uptake by Mucor hiemalis. Evelyn Balsano, Maranda Esterhuizen-Londt, Enamul Hoque, and Stephan Pflugmacher Journal of Applied Biology and Biotechnology, 2016, 4(4), 031-041 Doi: 10.7324/JABB.2016.40403 This work is licensed under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. CC BY-NC-SA 3.0 III) Responses of the antioxidative and biotransformation enzymes in the aquatic fungus Mucor hiemalis exposed to cyanotoxins. Evelyn Balsano, Maranda Esterhuizen-Londt, Enamul Hoque, and Stephan Pflugmacher Biotechnology Letters, 2017, 39(8), 1201-1209 Doi: 10.1007/s10529-017-2348-8 The final publication is available at Springer via http://dx.doi.org/10.1007/s10529-017-2348-8 III LIST OF CONFERENCES Toxin resistance in aquatic fungi poses environmentally friendly remediation possibilities: A study on the growth responses and biosorption potential of Mucor hiemalis EH5 against MC-LR, BMAA, and CYN. Evelyn Balsano, Maranda Esterhuizen-Londt, Enamul Hoque, and Stephan Pflugmacher Poster session presented at: FEMS 2015, 6th Congress of European Microbiologists 2015 Jun 7-11, Maastricht, The Netherlands. IV CONTENTS Acknowledgements ................................................................................................................... I List of manuscripts ................................................................................................................ III List of conferences ................................................................................................................. IV Contents .................................................................................................................................... V Figure Index ........................................................................................................................... IX Table Index .......................................................................................................................... XIII Abstract ................................................................................................................................. XV Zusammenfassung ............................................................................................................. XVII Abbreviations ...................................................................................................................... XIX 1 Introduction ........................................................................................................................ 1 1.1 Cyanotoxins .................................................................................................................. 2 1.2 Oxidative stress and antioxidant defensive system .................................................. 5 1.3 Cyanotoxin metabolism and detoxification in living organisms ............................. 8 1.4 Removal of cyanotoxins – Biodegradation .............................................................. 11 Mycoremediation ........................................................................................................ 13 1.5 Aims and objectives ................................................................................................... 17 2 Toxin resistance in aquatic fungi poses environmentally friendly remediation possibilities: A study on the growth responses and biosorption potential of Mucor hiemalis EH5 against cyanobacterial toxins ......................................................................... 21 Abstract ..................................................................................................................... 22 2.1 Introduction ............................................................................................................... 23 2.2 Materials and Methods ............................................................................................. 26 2.2.1 Fungal strain ................................................................................................... 26 2.2.2 Cyanobacterial toxins ..................................................................................... 26 2.2.3 Culture conditions ........................................................................................... 26 2.2.4 Disk diffusion assay: Inoculation and exposure ............................................. 26 2.2.5 Measurement of radial growth rate and biomass production .......................... 27 2.2.6 Biosorption: Exposure scenario and sample collection .................................. 27 2.2.7 Toxin extraction .............................................................................................. 28 V 2.2.8 LC-MS/MS ..................................................................................................... 29 2.2.9 Statistical analysis .......................................................................................... 30 2.3 Results and Discussion .............................................................................................