Monitoring of bacterial communities in a phytoremediation plant for the decontamination of polluted marine sediments

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 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 months after the construction of the treatment basin the original bacterial communities evolved into a single bacterial community, homogeneously distributed in the whole area. The bacterial community got stabilized after one year from the construction of the treatment basin. The phytoremediation process influenced the development of a specific bacterial community of the treatment basin that is completely different from the bacterial communities harbored by the basin at the beginning of the experimentation.