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Monitoring of Bacterial Communities in a Phytoremediation Plant for the Decontamination of Polluted Marine Sediments”

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Research Doctorate School in Biological and Molecular Sciences Cycle XXV “Monitoring of bacterial communities in a phytoremediation plant for the decontamination of polluted marine sediments” Candidate: Carolina Chiellini Supervisors: Dott. Giulio Petroni Prof. Ing. Renato Iannelli 2013 BIO/05 INDEX SUMMARY .......................................................................................................................................................... 1 RIASSUNTO ........................................................................................................................................................ 3 1 INTRODUCTION: AN OVERVIEW ON THE TOPIC ........................................................................... 5 1.1 THE NORMATIVE CONCERNING DREDGED SEDIMENTS ........................................................................ 7 1.1.1 The European scenario ......................................................................................................................... 7 1.1.2 The Italian scenario ................................................................................................................................ 9 1.2 THE PROBLEM OF DREDGED SEDIMENTS ................................................................................................ 12 1.2.1 Types and sources of pollutants in marine sediments .............................................................. 12 1.2.2 How to treat polluted sediments ....................................................................................................... 13 1.2.2.1 Chemical treatments .............................................................................................................. 14 1.2.2.2 Biological treatments ............................................................................................................. 17 1.3 PHYTOREMEDIATION .................................................................................................................................... 18 1.4 AIM OF THE STUDY ......................................................................................................................................... 20 1.5 REFERENCES .................................................................................................................................................... 21 2 MATERIALS AND METHODS USED TO STUDY BACTERIAL COMMUNITIES ................. 24 2.1 BIOMOLECULAR ANALYSIS TO STUDY MICROBIAL ECOLOGY ........................................................ 24 2.1.1 The Polymerase Chain Reaction ...................................................................................................... 26 2.1.2 Full Cycle rRNA approach ............................................................................................................... 27 2.1.3 Terminal Restriction fragment Length Polymorphism ............................................................ 29 2.2 STATISTICAL ANALYSIS APPLIED ON DATA OBTAINED FROM FINGERPRINT MOLECULAR METHODS ................................................................................................................................................................. 30 2.2.1 Cluster Analysis ..................................................................................................................................... 31 2.2.2 Nonmetric Multidimensional Scaling (NMDS) .......................................................................... 34 i 2.2.3 Diversity Indices .................................................................................................................................... 35 2.3 REFERENCES .................................................................................................................................................... 38 3 THE AGRIPORT PROJECT ....................................................................................................................... 44 3.1 ASSESSMENT OF POLLUTION IMPACT ON BIOLOGICAL ACTIVITY AND STRUCTURE OF SEABED BACTERIAL COMMUNITIES IN THE PORT OF LIVORNO (ITALY) ............................................................. 47 3.2 BACTERIAL COMMUNITIES IN POLLUTED SEABED SEDIMENTS: A MOLECULAR BIOLOGY ASSAY IN LEGHORN HARBOR ............................................................................................................................. 62 3.3 TEMPORAL CHARACTERIZATION OF BACTERIAL COMMUNITIES IN A PHYTOREMEDIATION PILOT PLANT AIMED AT DECONTAMINATING POLLUTED SEDIMENTS DREDGED FROM LEGHORN HARBOR, ITALY ...................................................................................................................................................... 86 4 OTHER STUDIES CONCERNING BACTERIAL COMMUNITIES COMPOSITION IN ENVIRONMENTAL MATRICES PERFORMED DURING THE PHD PERIOD ............................ 97 4.1 BIOFOULING OF REVERSE OSMOSIS MEMBRANES USED IN RIVER WATER PURIFICATION FOR DRINKING PURPOSES: ANALYSIS OF MICROBIAL POPULATIONS .............................................................. 98 4.2 CHARACTERIZATION AND COMPARISON OF BACTERIAL COMMUNITIES SELECTED IN CONVENTIONAL ACTIVATED SLUDGE AND MEMBRANE BIOREACTOR PILOT PLANTS: A FOCUS ON NITROSPIRA AND PLANCTOMYCETES BACTERIAL PHYLA ........................................................................ 115 4.3 EFFECTS OF OZONATION ON THE BACTERIAL COMMUNITIES COMPOSITION PRESENT IN A FULL SCALE PLANT FOR DOMESTIC WASTEWATER TREATMENT ......................................................... 147 5 ACKNOWLEDGMENTS .................................................................................................................................. ii Summary This thesis was performed within the “Agriport” European Project (Agricultural Reuse of Polluted Dredged Sediments, Eco-innovation EU Project n. ECO/08/239065). The Agriport project aims at developing a new technology for treating polluted sediments dredged from the seabed of a commercial port through phytoremediation. Thanks to plant activities and microorganism metabolism, it is possible to recover dredged saline sediments by decontaminating and conditioning them until the obtainment of an artificially prepared soil that is reusable in the terrestrial environment. This is an important advantage from the environmental point of view, and allows to partially solve one of the main problems of most commercial ports, that is the accumulation, storage and disposal of polluted dredged sediments. The study was divided into two main parts, involving the study of bacterial community in fresh marine sediments collected in the seabed of an industrial harbor, and the study of bacterial communities involved in the phytoremediation process of marine contaminated sediments. During the first part of the study, an accurate analysis on bacterial communities composition in fresh marine sediments, directly collected by a scuba diver in five sites of Leghorn harbor was performed. Aim of this first part of the study was to assess the impact of pollution on seabed bacterial diversity, structure and activity in the Port of Leghorn. Seabed sediments of commercial ports are often characterized by high levels of pollution, mainly represented by organic matter, heavy metals and hydrocarbons. One of the main characteristic of harbor sites is the removal of organic matter. Consumption capacity of organic matter is mainly due to the activity of microorganisms. The knowledge of bacterial communities dynamics in fresh marine sediments allowed us to compare the data with the results obtained in the second part of the study, involving bacterial community dynamics in the phytoremediation plant, that were monitored for two years. The phytoremediation plant was made up of a sealed 80 m3 basin that was filled with a mixture of dredged sediments (75%) and natural soil (25%). It was planted with three plant species, and has been properly cultivated with optimized fertilization and irrigation for two years. The techniques developed and used in this thesis were Terminal Restriction Fragment Length Polymorphism (T-RFLP) followed by appropriate statistical analysis, and construction of 16S rRNA clone libraries. Results of the first part of the study, focusing on freshly collected marine sediments, showed that the industrial harbor is mainly contaminated by variable levels of petroleum hydrocarbons and heavy metals, which affect the structure and activity of the bacterial population. A stimulatory effect of nutrients on biological activities and bacterial communities was clearly observed. A 1 stimulation of bacterial development driven by petroleum hydrocarbon and heavy metals was also detected, although with less evidence. Library data, phylogenetic analysis, and T-RFLP coupled with in silico digestion of the obtained sequences, evidenced the dominance of Proteobacteria and the high percentage of Bacteroidetes in all sites. The approach highlighted similar bacterial community among samples coming from the five sites, suggesting a modest differentiation among bacterial communities of different harbor seabed sediments, and hence the capacity of bacterial communities to adapt to different levels and types of pollution. The second part of the study, focusing on bacterial community dynamics in the phytoremediation plant, highlighted that, at the beginning of the experimentation (construction of the treatment basin), each component used to assemble the phytoremediation treatment basin was characterized by its own bacterial community which was differentiated from the others. Six
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