Master’s dissertation submitted in partial fulfilment of the requirements for the joint degree of International Master of Science in Environmental Technology and Engineering an Erasmus+: Erasmus Mundus Master Course jointly organized by Ghent University, Belgium University of Chemical Technology, Prague, Czech Republic UNESCO-IHE Institute for Water Education, Delft, the Netherlands Academic year 2014 – 2015. Induction of biphenyl degradation pathway genes by plant secondary metabolites Host University: University of Chemical Technology, Prague, Czech Republic Binyam Woldehawariat Promotor: Assoc. Prof. Ondřej Uhlík, MSc., Ph.D. This thesis was elaborated at University of Chemical Technology, Prague, Czech Republic and defended at University of Chemical Technology, Prague, Czech Republic within the framework of the European Erasmus Mundus Programme “Erasmus Mundus International Master of Science in Environmental Technology and Engineering " (Course N° 2011-0172) © 2015 Prague, Czech Republic, Binyam Woldehawariat, Ghent University, all rights reserved. ii Deze pagina is niet beschikbaar omdat ze persoonsgegevens bevat. Universiteitsbibliotheek Gent, 2021. This page is not available because it contains personal information. Ghent Universit , Librar , 2021. DECLARATION This thesis/dissertation was written at the Department of Biochemistry and Microbiology of the University of Chemical Technology, Prague, Czech Republic from February to August 2015. I hereby declare that this thesis is my own work. Where other sources of information have been used, they have been acknowledged and referenced in the list of used literature and other sources. I have been informed that the rights and obligations implied by Act No. 121/2000 Coll. on Copyright, Rights Related to Copyright and on the Amendment of Certain Laws (Copyright Act) apply to my work. In particular, I am aware of the fact that the University of Chemical Technology in Prague has the right to sign a license agreement for use of this work as school work under §60 paragraph 1 of the Copyright Act. I have also been informed that in the case that this work will be used by myself or that a license will be granted for its usage by another entity, the Institute of Chemical Technology in Prague is entitled to require from me a reasonable contribution to cover the costs incurred in the creation of the work, according to the circumstances up to the full amount. I agree to the publication of my work in accordance with Act No. 111/1998 Coll. on Higher Education and the amendment of related laws (Higher Education Act). In Prague on August 12, 2015 iv Acknowledgements I would like to thank Assoc. Prof. Ondřej Uhlík for his professional guidance, support and patience throughout this diploma thesis and beyond. I am indebted for his helpful suggestions during the course of my research. His advice and expertise were invaluable to accomplish my diploma thesis. I would like to express my deepest appreciation to my tutor, Michal Strejček, MSc. for his time, patience and all the support he offered me in realizing my diploma thesis. He went beyond in helping and guiding me through the most important and difficult part of my research work. Special thanks to Lucie Musilová for all the support you gave me to accomplish this work. I am also grateful to Eglantina, Serena, Jáchym, Honza and the rest, for the support, advice and comforting thoughts in these past 6 months. My heartfelt gratitude to the IMETE Management Board for making IMETE program possible. Thank you to Ineke Melis of UNESCO-IHE, Ing. Jana Bartáčková, Ph.D. of ICTP and Evelien Vandevelde of Ghent for handling all arrangements and support in Delft, Prague and Ghent. Special gratitude to European Commission for sponsoring my study with Erasmus Mundus scholarship. Finally, I would like to thank and give special tribute to my wife, Sossena whose constant moral and emotional support has guided me to reach at this stage of my career. Thanks also to my mother and my friends for always being there for me and encouraged me to keep on with my dreams. v Abstract Many previous studies have reported that polychlorinated biphenyls (PCBs) are degraded more efficiently in vegetated soils when compared to non-vegetated soils. These findings led to the hypothesis that the enhancement in degradation could be attributed to the presence of plant secondary metabolites (PSMs) in vegetated soils, released by plant roots. Recent laboratory studies have also demonstrated that PSMs may serve as natural substrates and/or inducers for the biphenyl catabolic pathway. Thus, the main objective of the present study was to investigate biphenyl catabolic pathway induction potential of selected PSMs in four bacterial strains: Achromobacter denitrificans AD400, Pseudomonas alcaliphila JAB1, Achromobacter xylosoxidans S3, and Pseudomonas putida S9. By identifying PSMs that could be used instead of biphenyl, we hope to contribute in the development of new approaches for bioremediation of PCB-contaminated soils. In the present study, all the strains were grown co-metabolically on sodium pyruvate plus one of the 14 different PSMs investigated. We confirmed the induction of the biphenyl catabolic pathway by the PSMs using quantitative real-time polymerase chain reaction (RT-qPCR) of the bphA gene. A mathematical model from literature was used to quantify the relative expression of bphA gene with respect to the control (sodium pyruvate). The RT-qPCR results showed that for strains JAB1 and S3 all the PSMs were able to induce the bphA gene, whereas in strain AD400 all except p-cymene were able to induce bphA. However, none of the PSMs were able to induce bphA in strain S9. The investigation of the relative quantification of bphA gene showed that a number of the PSMs tested were able to induce the bphA significantly higher (p < 0.05) than the control. Additionally, in strain AD400, coumarin induced bphA gene significantly higher (p < 0.05) than biphenyl, whereas in strain JAB1 ferulic acid and p-cymene were able to induce bphA gene significantly higher (p < 0.05) than biphenyl itself. Naringin, α-pinene, p-hydroxybenzoic acid, vanillic acid, caffiec acid, limonene, and carvone are other significant inducers identified in the present study. In conclusion, the present study demonstrated that a number of PSMs have the potential to induce the biphenyl catabolic pathway at the same level or even higher than biphenyl itself. Thus, considering the fact that some PCBs can also be degraded by the same catabolic pathway, PSMs have the potential to replace biphenyl as a proper inducer for the bioremediation of PCB- contaminated sites. vi Table of Contents Acknowledgements ................................................................................................................................. v Abstract .................................................................................................................................................. vi List of Figures ........................................................................................................................................ ix List of Tables .......................................................................................................................................... x Abbreviations ......................................................................................................................................... xi Chapter 1: Introduction ........................................................................................................................... 1 Chapter 2: Literature review ................................................................................................................... 2 2.1 Polychlorinated biphenyls (PCBs) ................................................................................................ 2 2.2 Environmental fate of PCBs ......................................................................................................... 3 2.3 Potential adverse effects of PCBs ................................................................................................. 4 2.4 Remediation techniques of PCB-contaminated soils .................................................................... 4 2.4.1 Physicochemical methods ...................................................................................................... 4 2.4.2 Bioremediation ....................................................................................................................... 6 2.4.3. Plant secondary metabolites .................................................................................................. 9 2.4.4. Rhizodegradation ................................................................................................................ 10 Chapter 3: Objectives ............................................................................................................................ 12 Chapter 4: Material and Methods.......................................................................................................... 13 4.1 Materials ..................................................................................................................................... 13 4.1.1 Chemicals ............................................................................................................................. 13 4.1.2 Primers, enzymes and commercial kits ................................................................................ 13 4.1.3 Bacterial