University of Thessaly Department of Biochemistry and Biotechnology Isolation of bacteria that degrade carbamate insecticides and characterization of the functional and ecological role of bacterial genes involved in their hydrolysis in soil A thesis submitted by Konstantina Rousidou For the degree of Doctor of Philosophy 2020 Institutional Repository - Library & Information Centre - University of Thessaly 07/10/2021 11:56:00 EEST - 170.106.33.180 Isolation of bacteria that degrade carbamate insecticides and characterization of the functional and ecological role of bacterial genes involved in their hydrolysis in soil 2 Institutional Repository - Library & Information Centre - University of Thessaly 07/10/2021 11:56:00 EEST - 170.106.33.180 Members of the Examination Committee • Professor D. G. Karpouzas (Environmental Microbiology and Biotechnology), Department of Biochemistry and Biotechnology of University of Thessaly, Supervisor and Member of the Advisory Committee • Associate Professor K. Papadopoulou (Plant Biotechnology), Department of Biochemistry and Biotechnology of University of Thessaly, Member of the Advisory Committee • Associate Professor G. Tsiamis (Environmental Microbiology), Department of Environmental Engineering of University of Patras. Member of the Advisory Committee • Professor C. Ehaliotis (Soil Fertility and Biology), Department of Natural Resources Management and Agricultural Engineering of Agricultural University of Athens • Dr. G. Nicol, Environmental Microbial Genomics Group, University of Lyon • Associate Professor S. Ntougias (Environmental Microbiology), Department of Environmental Engineering of Democritus University of Thrace • Professor T. Vogel, Environmental Microbial Genomics Group, University of Lyon 3 Institutional Repository - Library & Information Centre - University of Thessaly 07/10/2021 11:56:00 EEST - 170.106.33.180 Konstantina Rousidou Isolation of bacteria that degrade carbamate insecticides and characterization of the functional and ecological role of bacterial genes involved in their hydrolysis in soil 4 Institutional Repository - Library & Information Centre - University of Thessaly 07/10/2021 11:56:00 EEST - 170.106.33.180 To my family 5 Institutional Repository - Library & Information Centre - University of Thessaly 07/10/2021 11:56:00 EEST - 170.106.33.180 A Ship in Harbor Is Safe, But that Is Not What Ships Are Built For John A. Shedd 6 Institutional Repository - Library & Information Centre - University of Thessaly 07/10/2021 11:56:00 EEST - 170.106.33.180 Acknowledgements Firstly, I would like to express my sincere gratitude to my supervisor, Professor Karpouzas Dimitrios, for his continuous support during my Ph.D. study and related research. I am grateful for his motivation, understanding and patience throughout this long walk with all its ‘’ups and downs’’. His scientific guidance helped me in all the time of research and writing of this thesis. I consider myself to be privileged to have been his student. I would, also, like to extend my gratitude to Associate Professor Papadopoulou Kalliope for taking the time to discuss and give helpful advice whenever needed. Her support has been of great importance to me. Additionally, I would like to thank Associate Professor Tsiamis George for serving as one of my advisory committee members. Furthermore, I would like to thank Professor Ehaliotis Constantinos (special thanks, also, for his encouragement when Ι needed it), Dr. Graeme Nicol, Associate Professor Ntougias Spyridon and Professor Vogel Timothy for accepting to be members of my examination committee. Thanks, should also go to people who have contributed in some parts of this work: M.Sc. Chanika Eleni, M.Sc. Georgiadou Dafne, M.Sc. Karaiskos Dionysis, M.Sc. Despoina Myti, M.Sc. Kolovos Panagiotis and Dr. Tourna Maria. I would, of course, like to thank the past and present lab members Dr. Papadopoulou Evangelia, Dr. Perruchon Chiara, Dr. Karas Panagiotis, Dr. Katsoula Athanasia, Dr. Vasileiadis Sotiris, Dr. Skiada Vasiliki, Assistant Professor Tsikou Daniela, Dr. Garagounis Constantine, Dr. Krokida Afrodite, Dr. Katsarou Dimitra and Dr. Stedel Catalina for our excellent cooperation and friendship all these years. Last but not least, I would like to thank my family for providing me with constant support and continuous encouragement throughout my years of study and through the process of researching and writing this thesis. 7 Institutional Repository - Library & Information Centre - University of Thessaly 07/10/2021 11:56:00 EEST - 170.106.33.180 Table of contents Acknowledgements ___________________________________________________ 6 Abstract ___________________________________________________________ 11 Περίληψη __________________________________________________________ 14 CHAPTER 1 General Introduction ___________________________________ 17 1.1 Pesticides And Pesticides Use ___________________________________ 18 1.2 Environmental Fate of Pesticides ________________________________ 19 1.2.1 Processes Responsible for the Accumulation of Pesticides in Soil: Adsorption- Desorption ___________________________________________________ 20 1.2.2 Processes Responsible for Pesticides Transportation in Soil _____________ 21 1.2.3 Processes Responsible for Pesticides Disappearance in Soil _____________ 23 1.3 Biodegradation of Pesticides in Soil ______________________________ 25 1.3.1 Microorganisms Involved in the Biodegradation of Pesticides ___________ 26 1.3.2 Microbial Adaptation to Pesticides _________________________________ 27 1.3.3 Mechanisms of Pesticide Biodegradation ____________________________ 29 1.3.4 Microbial Enzymes Involved in Pesticides Biodegradation ______________ 30 1.3.5 Factors Influencing the Biodegradation of Pesticides in Soils ____________ 32 1.4 Classification of Pesticides ______________________________________ 33 1.4.1 Classification by Mode of Action __________________________________ 34 1.4.2 Classification by Target Pest ______________________________________ 34 1.4.3 Classification by Chemical Structure _______________________________ 34 1.5 Main Insecticide Groups _______________________________________ 37 1.6 Carbamates __________________________________________________ 38 1.7 Carbamate Insecticides ________________________________________ 40 1.7.1 History and Classification of Carbamates Insecticides __________________ 40 1.7.2 Mode of Action and Toxicity _____________________________________ 41 1.7.3 Environmental Fate _____________________________________________ 42 1.7.4 Biodegradation ________________________________________________ 44 1.8 Oxamyl ______________________________________________________ 46 1.8.1 Physicochemical Properties and Uses _______________________________ 46 1.8.2 Environmental Fate _____________________________________________ 47 1.8.3 Biodegradation ________________________________________________ 50 1.8.4 Toxicity ______________________________________________________ 51 8 Institutional Repository - Library & Information Centre - University of Thessaly 07/10/2021 11:56:00 EEST - 170.106.33.180 1.9 Carbofuran __________________________________________________ 52 1.9.1 Physicochemical Properties and Uses _______________________________ 52 1.9.2 Environmental Fate _____________________________________________ 53 1.9.3 Biodegradation ________________________________________________ 54 1.9.4 Toxicity ______________________________________________________ 55 1.10 AIMS OF THE THESIS _______________________________________ 56 1.11 References ___________________________________________________ 57 CHAPTER 2 Isolation of oxamyl-degrading baceria and identification of cehA as a novel oxamyl hydrolase gene ______________________________________ 73 2.1 Introduction _________________________________________________ 74 2.2 Materials and Methods ________________________________________ 76 2.2.1 Pesticides and Media ____________________________________________ 76 2.2.2 Soil Microbial Degradation of Oxamyl and Oxamyl Oxime _____________ 76 2.2.3 Isolation of Oxamyl-degrading Bacteria _____________________________ 77 2.2.4 Phylogenetic Classification of the Oxamyl-degrading Bacteria ___________ 77 2.2.5 Growth Kinetics and Oxamyl Degradation ___________________________ 80 2.2.6 Mineralization of 14C-labelled Oxamyl by the Oxamyl-degrading Bacteria _ 80 2.2.7 Degradation of Other Pesticides by the Oxamyl-degrading Bacterium OXA20 ____________________________________________________________ 81 2.2.8 Detection and Isolation of a Carbamate-Hydrolase Gene ________________ 81 2.2.9 Transcription Analysis of the cehA Gene in Oxamyl-degrading Bacteria ___ 84 2.2.10 Utilization of Methylamine by Oxamyl-degrading Isolates ______________ 85 2.2.11 Pesticides Analysis _____________________________________________ 85 2.3 Results ______________________________________________________ 86 2.3.1 Degradation of Oxamyl and Oxamyl Oxime in Soil and Enrichment Cultures ____________________________________________________________ 86 2.3.2 Isolation and Phylogenetic Classification of the Oxamyl-degrading Isolates 89 2.3.3 Growth Kinetics and Hydrolysis of Oxamyl __________________________ 89 2.3.4 Mineralization of 14C-labelled Oxamyl by the Isolated Bacteria __________ 92 2.3.5 Degradation of Other Pesticides by the Oxamyl-degrading Strain OXA20 __ 94 2.3.6 Detection of a Carbamate-Hydrolase Gene in the Oxamyl-degrading Bacteria _____________________________________________________ 95 2.3.7 Transcription Analysis of the cehA Gene in Oxamyl-degrading Bacteria