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Oil Spills Bioremediation in Marine Environment-Biofilm Characterization Around Oil Droplets

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Oil Spills Bioremediation in Marine Environment-Biofilm Characterization Around Oil Droplets Oil spills bioremediation in marine environment-biofilm characterization around oil droplets Βιοαποδόμηση πετρελαιοειδών σε θαλάσσιο περιβάλλον - Χαρακτηρισμός σχηματισμού βιοστιβάδας σε σταγονίδια υδρογονανθράκων i ii Examination Committee Nicolas Kalogerakis – Supervisor Professor, Division II: Environmental Process Design and Analysis, School of Environmental Engineering, Technical University of Crete, Chania, Greece Nikos Pasadakis – Advisory Committee Associate Professor School of Mineral Resources Engineering, Technical University of Crete, Chania, Greece Dr. Thomas R. Neu – Advisory Committee Head of Group - Microbiology of Interfaces, Department River Ecology, Helmholtz Centre for Environmental Research – UFZ, Magdeburg, Germany Evangelos Gidarakos Professor, Division I: Environmental Management School of Environmental Engineering, Technical University of Crete, Chania, Greece Elia Psillakis Associate Professor, Division II: Environmental Process Design and Analysis School of Environmental Engineering, Technical University of Crete, Chania, Greece Danae Venieri Assistant Professor, Division II: Environmental Process Design and Analysis School of Environmental Engineering, Technical University of Crete, Chania, Greece Marina Pantazidou Associate Professor Department of Geotechnical Engineering, School of Civil Engineering, National Technical University of Athens, Athens, Greece iii iv Acknowledgments This work was funded by FP-7 PROJECT No. 266473 “Unravelling and exploiting Mediterranean Sea microbial diversity and ecology for xenobiotics’ and pollutants’ clean up” – ULIXES and by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: Heracleitus II. Investing in knowledge society through the European Social Fund. First I would like to express my gratitude to my supervisor professor N. Kalogerakis for his support, advice and guidance in completing this study. His judgment, frustrating but also creative, was a stimulating process throughout all these years. Special thanks to Associate Professor N. Pasadakis for his guidance and valuable advice during my work in his lab and for the long, stimulating though, discussions about this work. I sincerely thank Dr. T. Neu for his hospitality, guidance and valuable observations during my work in his lab at UFZ, Magdeburg. At this point I would also like to express special thanks to his assistant Ute Kuhlicke for her support, instructions but most of all for her company during the long dark hours spent in front of the confocal laser scanning microscope (CLSM). I also would like to thank Assistant Professor D. Venieri for her valuable advice, kind support during my work and particularly for her consult in microbiological and molecular assays whenever needed. I have also to acknowledge S. Fodelianakis for his contribution in Pyrotag sequencing analysis of this work. I have to acknowledge the seven-member Examination Committee for their agreement to participate and contribute with comments to this PhD thesis. I must extend a special word of Thanks to friends and family who have supported me throughout my various tasks of this PhD thesis. Thank you Arly, Dora, Magda for all the laughs and keeping me sane in the lab and encouraging me to carry on throughout disappointing and difficult periods. Last but certainly not least to my parents Panagiotis and Eleni for being there for me throughout my study period supporting my choices and encouraging me to follow my decisions respecting my values no matter how difficult they would be. I would like to thank my lovely husband George for his support and understanding, although he did not always like the time I spent on this work, I know his is proud on the end result. v vi Abstract Oil spills are considered a widespread problem after the last accident at the Mexico Gulf that poses great threat for any ecosystem. Specifically Mediterranean coastal regions are exposed to oil pollution due to extensive industrialization and urbanization and transport of crude and refined oil to and from refineries. The impact of an oil spill in this closed sea and particularly in the Greek coastline and sea that are popular touristic destinations and shelter of many marine species can be disastrous. First response options, such as physical removal (e.g. booms, skimmers, adsorbents, etc.) and dispersants whenever applicable due to their potential toxicity rarely achieve complete cleanup of oil spills. Nevertheless, major oil spills highlight the need for environmentally responsible and cost-effective mitigation technologies. Bioremediation through bioaugmentation (addition of oil-degrading bacteria) and/or biostimulation (addition of nutrients N&P) constitutes a promising strategy for combatting oil spills following first response actions and recent technological advances could promote bioremediation to a priority option in combating oil spills. However, bioaugmentation is one of the most controversial issues in bioremediation since nutrient addition alone has been found to have a greater effect on oil biodegradation than the addition of microbial products that are highly dependent on environmental conditions. There is increasing evidence that the best way to overcome the above barriers is to exclusively use microorganisms indigenous to the sites (soil, sand, and water) to be decontaminated, an approach termed “autochthonous bioaugmentation” (ABA). The specific aims of the present work were to investigate possible methods to enhance the rate of biodegradation of oil in a contaminated marine environment (both seawater and shoreline). Hence we investigated the capability of either acclimated indigenous microbial consortium or hydrocarbon degraders consortium enriched from seawater samples taken from Hellenic Petroleum Refinery (Athens, Greece) a site exposed to chronic pollution with crude oil (ABA) in the presence or absence of other rate limiting factors like nutrients and biosurfactants (biostimulation) as a potential strategy for the successful remediation of polluted marine environments. In addition the effectiveness of these certain acclimated consortia (ABA) was compared to indigenous population activity (biostimulation) on the bioremediation of oil spills. Specifically the effects of the lipophilic nutrients (uric acid and lecithin) and inorganic nutrients (KNO3, K2HPO4) with or without biosurfactants on the degradation of crude oil hydrocarbons in both seawater and sand matrix were also examined. While bioremediation in liquid matrices (seawater) is implemented in a more direct way, in the soil matrix (sand) is achieved through landfarming which is both simple and cost- effective to implement compared with other treatment technologies. Thus the outcome approaches include 4 sets of experiments: 1. Autochthonous bioaugmentation and/or biostimulation of seawater microcosm (i.e. Seawater 1) 2. Autochthonous bioaugmentation & biostimulation with isolated hydrocarbon degraders consortium of seawater microcosm (i.e. Seawater 2) 3. Landfarming of oil polluted beach sand through biostimulation (i.e. Sand 1) 4. Landfarming of oil polluted beach sand through autochthonous bioaugmentation & biostimulation (i.e. Sand 2) The method which has been used for the evaluation of these bioremediation methods is based on a modified bioremediation agent effectiveness testing protocol by EPA vii (40 CFR Ch. I, Pt 300, App. C, 2003). The protocol tests for microbial activity by Most Probable Number (MPN) determination and quantifies the disappearance of saturated hydrocarbons and polynuclear aromatic hydrocarbons (PAHs) by GC-MS analysis. Moreover the disappearance extent (degradation rate) of certain compounds from both saturated fraction (n-alkanes) and aromatic fraction (PAHs was also investigated through biodegradation kinetics analysis (Batch reactor). Furthermore identification of the key microorganisms in oil biodegradation community response and composition changes among different amendments was also performed through molecular analysis (PCR, RT-PCR, pyrotag Sequencing) of DNA extracts from each treatment. Hydrocarbon degrading bacteria as shown by several studies have a high affinity to oil droplets which indicates that oil bioremediation is a complex process that involves interactions between oil and microorganisms under certain environmental conditions. Therefore in this study another set of experiments investigating and characterizing the interaction of hydrocarbon degraders consortia on oil and eicosane droplets (cellular level) by means of confocal laser scanning microscopy (CLSM) was also included. Moreover their response after the addition of certain commercial dispersants just like those that have been or could be used in the event of a real oil spill (Corexit, S200, Marichem) but also of more environmentally friendly biosurfactants (Rhamnolipids) was also examined. Investigation through chemical, microbiological and kinetics analysis (specific degradation rate, qs) has revealed that Seawater 1 experimental set in which acclimated or not indigenous microorganisms were used performed far better than Sand 1 experimental set in which degradation was induced only by indigenous microbial populations and despite the fact that combined Rhamnolipids and lipophilic nutrients were used still microbial response was not as strong as in Seawater 1 experimental set. Bioavailability of oil hydrocarbons is the critical
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