University of Thessaly Department of Biochemistry and Biotechnology Isolation and study of bacteria able to degrade pesticides contained in the wastewaters produced by the fruit-packaging industry A thesis submitted by Chiara Perruchon For the degree of Doctor of Philosophy 2014 Institutional Repository - Library & Information Centre - University of Thessaly 09/12/2017 02:48:33 EET - 137.108.70.7 Members of the Examination Committee Assistant Prof. Dimitrios Karpouzas (Environmental Microbiology and Biotechnology), University of Thessaly, Department of Biochemistry and Biotechnology, Supervisor and Member of the Advisory Committee Associate Prof. Konstantinos Ehaliotis (Soil Fertility and Biology), Agricultural University of Athens, Department of Natural Resources Management and Agricultural Engineering, Member of the Advisory Committee Associate Prof. Kalliope Papadopoulou (Plant Biotechnology), University of Thessaly, Department of Biochemistry and Biotechnology, Member of the Advisory Committee Associate Prof. Konstantinos Kormas (Ecology of Aquatic Microorganisms), University of Thessaly, School of Agriculture Prof. Urania Menkissoglou-Spiroudi (Pesticide-Chemical Synthesis and Activity), Aristotle University of Thessaloniki, School of Agriculture Assistant Prof. Dimitrios Mossialos (Microbial Biotechnology), University of Thessaly, Department of Biochemistry and Biotechnology Assistant Prof. Spyridon Ntougias (Environmental Microbiology), Democritus University of Thrace, Department of Environmental Engineering Institutional Repository - Library & Information Centre - University of Thessaly 09/12/2017 02:48:33 EET - 137.108.70.7 Abstract Fruits are particularly vulnerable to fungal infestations or other physiological disorders during storage thus diminishing their market value. Post-harvest fruit treatments with fungicides like thiabendazole (TBZ), imazalil (IMZ), and ortho-phenyl-phenol (OPP), and antioxidants like diphenylamine (DPA) are the most effective means to minimize fruit spoilage in storage. The latter is no more registered for use in the EU, although exemption authorizations for 120 days have been given considering that there are no equally effective alternatives in the market for the control of apple scald. Pesticide application methods in fruit-packaging plants (dipping, drenching, spraying, or waxing) are water-based generating at the end of the process large volumes of wastewaters containing high concentrations of pesticide (0.2 to 2 g/l depending on the molecule). Direct discharge of these wastewaters into the environment without prior treatment constitutes a serious point-source of contamination. This problem becomes more acute when the high persistence (TBZ, IMZ) and toxicity (all) of the pesticides used is taken in account. Therefore, a wastewater treatment system able to efficiently depurate the wastewaters derived from the post-harvest handling of fruits is required, considering also that authorization for the use of those pesticides TBZ, IMZ, OPP, and DPA was granted under the clause that an effective treatment of the wastewaters produced is operative. However, the only system actually patented (CONTROL TEC-ECO® system) is highly costly precluding its market uptake. Thus, a sustainable, cheap, effective, and environmentally friendly method for the depuration of the fruit packaging wastewater is needed. Microbial degradation of pesticide is considered the most important process for their environmental loss. The development of biological treatment facilities based on the degrading ability of microorganisms seems promising. The implementation of such systems will require inoculation with start-up inocula possessing high degradation capacity against the target compounds. However, very little (OPP, DPA) or nothing (TBZ, IMZ) is known regarding the microbial degradation of those chemicals. The current doctoral thesis aimed to fulfill this gap and provide novel insights into the microbial degradation of those pesticides with future practical implications for environmental protection. Thus the main research aim of this thesis was to isolate and characterize bacteria able to degrade the major pesticides used in the fruit packaging industry (TBZ, IMZ, OPP, and DPA). Enrichment cultures from soils collected from wastewater disposal sites led to the isolation of TBZ, OPP, and DPA-degrading bacteria, while, despite different attempts, it was not possible to isolate any bacterium able to degrade IMZ. Three TBZ-degrading consortia able to rapidly degrade the fungicide and use it as a C and N source were obtained. DGGE analysis and cloning showed that all TBZ-degrading Institutional Repository - Library & Information Centre - University of Thessaly 09/12/2017 02:48:33 EET - 137.108.70.7 consortia were composed of two to eight main members of proteobacteria. Different microbiological (antibiotics treatment) and molecular (DNA/RNA-DGGE, cloning, q-PCR) approaches, showed that a Sphingomonas sp. strain was responsible for the degradation of TBZ, while the role of other bacterial members was probably supportive regarding nutrition or degradation of metabolites produced (C1-compounds). However, the bacterium was not obtained in pure culture due to its limited capacity to grow on the agar media tested. The TBZ-degrading consortium was able to degrade 750 mg/l TBZ in liquid culture and 500 mg/kg TBZ in artificially contaminated soil. Moreover, it was able to degrade TBZ in a wide range of pH (4.5- 7.5) and temperatures (15-37oC) and in the presence of OPP and DPA and their respective degrading bacteria. An OPP-degrading bacterium identified as S. haloaromaticamans was isolated in pure culture. The bacterium was able to rapidly degrade OPP only when supplemented with casamino acids or in the co-presence of at least another bacterial strain (e.g. Pseudomonas stutzeri), an observation suggesting the incapacity of the degrading isolate to synthesize some essential amino acids like the ones included in casamino acids. The isolated S. haloaromaticamans strain was able to metabolize up to 150 mg/l OPP within 7 days and to use it as an energy source for growth. OPP was metabolized by the isolate in a wide range of pH (4.5-9) and temperatures (4- 37oC) and in the presence of different pesticides (TBZ and DPA) used in the fruit packaging industry and of their pesticide-degrading microbes. Regarding DPA, a P. monteilii was isolated which was able to degrade rapidly DPA and use it as a carbon and nitrogen source. Molecular analysis showed that the specific strain had polymorphisms in the different copies of its 16S rRNA gene, a common feature of many Pseudomonads. The isolated bacterium was able to rapidly degrade spillage level concentrations of DPA in liquid media (2 g/l) and in artificially contaminated soil (1 g/kg). Its degrading activity was not impaired in a wide range of pH (4.5-9) and temperatures (4-37oC) or by the co- presence of other pesticides possibly contained in wastewaters (TBZ and OPP) and their respective degrading bacteria. Overall, the results of this thesis provide novel insights into the largely unknown area of microbial degradation of the pesticides contained in wastewaters from the fruit-packaging industry. The pesticide-degrading bacteria isolated showed promising properties which could make them valuable tools in future biotechnological applications including their use a) as tailor- made inocula in biological wastewater treatment facilities, b) in in situ bioaugmentation of pesticide-polluted wastewater disposal sites adjacent to fruit-packaging plants and c) for bioaugmentation of modified biobed systems receiving wastewaters from the fruit packaging industry. Follow up studies will focus on both basic and practical challenges that have arisen Institutional Repository - Library & Information Centre - University of Thessaly 09/12/2017 02:48:33 EET - 137.108.70.7 including a) the elucidation of the metabolic pathway via advanced analytical tools and of the genetic mechanisms involved in the degradation of the pesticides studied via comparative genomic – proteomic analysis and b) the full-scale application of the isolated bacteria in biological wastewater treatment facilities. Institutional Repository - Library & Information Centre - University of Thessaly 09/12/2017 02:48:33 EET - 137.108.70.7 Acknowledgements Thanks to all the members of the Examination Committee. Thanks a lot to my supervisor, Prof. Karpouzas (Prof!), for being my lighthouse in all these years. Thank you for the great opportunity you gave me. Thank you for your guidance, your constant presence, and your time. Thank you for your efforts to make me better. Thanks to all the friends of the Lab, the past and the present ones, to make it a nice place to go every morning: Afroditi, Daniela, Dimitra, Ioanna M., Ioanna P., Vasiliki, and to all the people that worked there, who for more, who for less time. A special thanks to Panito, Kona, and Euaggelia for the laughs, the advices, the help, the patience, the friendship. Thank you. Thanks to the students who accompanied me in some parts of this work: Tasos, Vasia, Stelios, Nadia, Xristos. Another special thanks to my “bioinformatic guru” Sotos: thanks for the help in the phylogenetic trees, but most of all thank you for your time, for your enthusiasm in teaching and in explaining me things. Thanks to Mr. Antonios and Ms. Nasia to welcoming me into their family and always making me feeling at home. Thanks to all my Greek friends without whom these years would not be so beautiful, in