Comunita' bentoniche sublitorali di batteri e microbi eucariotici: caratterizzazione e analisi comparativa lungo un gradiente antropico

More than 70% of the world population is spread over catchments draining inorganic substances and nutrients towards the coasts. This could cause and/or enforce eutrophication, which involves significant changes in the biochemical composition of organic sediment that affect benthic organisms altering their metabolic processes and changing community structure, biodiversity, and the relationships among species and trophic groups. Autotrophic and heterotrophic prokaryotic and eukaryotic microorganisms represent a major component of marine sediments in terms of biomass and productivity, and the possibility of using rapid, reproducible, and standardized molecular methods for the characterization of their genetic diversity encourages their employment as indicators of health of ecosystems. A greater understanding of these communities would be allowed studying them simultaneously with the communities of higher trophic levels, for which the responses to stresses have been more investigated and understood. Very few studies have done comparative observation of marine communities with different ecological functions but subjected to the same environmental conditions and to the same kind of stress. Therefore, at present there is the lack of an holistic approach, which is able to highlight at the same time the responses of different components of the food web. The aim of this thesis is to perform the characterization and analysis of the bacterial community and the community of eukaryotic microbes in order to comparatively analyze data with the ones provided by the Section of Marine Biology on the composition of the meiozoobenthos and macrozoobenthos communities, sampled simultaneously in the same area of study; for the first time with this study, community of four different levels are analyzed comparatively. Furthermore, two molecular fingerprinting techniques with different resolution power have been used for the characterization of microbial communities, one of which was refined and applied for the first time to the eukaryotic microbial community. The stress condition was detected in corrispondence to the sea intake area of “fiume Morto”, a channel that carries treated wastewater and direct discharges of the municipal territory placed on the north of the Arno river. Bathing it's permanently forbidden in this coastal area. The sediment sampling was performed at about 150 meters from the coast, identifying, in addition to the area impacted, two control areas on the south of this. In each area three sites were randomly selected, and two few meters detached replicas in each site. For each replica two consecutive samples were taken with the use of a bucket type Ekmann-Birge: the first in order to collect sediment for analysis bacterial community, the second to collect sediment for analysis of eukaryotic communities. Further collection of material were done in the same sites for the observation of meio and macrobenthic communities, performed in the laboratories of the section of marine biology. Moreover, collection of environmental parameters, such as the surface salinity, the depth, the geographical coordinates, the particle size of the substrate and the amount of organic matter, was made in each site. The techniques employed for the molecular characterization of microbial communities have been the Terminal Restriction Fragment Length Polymorphism (T-RFLP), for the investigation of sequence polymorphisms, separately performed using two restriction enzymes (AluI and BsuRI) with different cutting sequences, and Lenght Heterogeneity Polymerase Chain Reaction (LH-PCR), for the investigation of the length polymorphisms due to insertion or deletion of bases in the variable regions of the DNA, both of them applied to the gene encoding the 16S rRNA for the characterization of bacterial communities and to the gene encoding the 18S rRNA for the characterization of the communities of eukaryotic microbes. In particular, the LH-PCR technique was applied on region V1 of both genes, and in addition on region V4 just for eukaryotic microbes. The data were analyzed with the use of two different software: PAST and PRIMER. For microbial communities the number of Operational Taxonomic Unit (OTU), and the diversity indices of Shannon-Wiener and Simpson, were calculated for each sample. For all the communities the distribution of the samples was represented on a two-dimensional plane through nmMDS (non-metric multidimensional scaling), checking the significance of the groupings identified with the statistical test ANOSIM, and the comparison was made by means of the test RELATE. Using the BIO-ENV test, the distribution of all the communities has been linked to the distribution of the values of the environmental parameters. Microbial communities characterized in this study were found to be very diversified and characterized by an equal distribution as regards the abundances of the numerous OTU. For the eukaryotic microbial community a certain degree of sensitivity to the gradient has emerged, although it was not highlighted by any significant correlation with the measured environmental parameters, and a weak correlation with the communities of the upper levels was detected, especially between the data obtained with the LH-PCR for region V1 and macrofauna and between those obtained with the T-RFLP using the enzyme AluI and meiofauna. This has not happened for the bacterial community. These weak correlations may indicate that the identified stress was indeed not strong enough to cause obvious changes in the microbial communities. The use of multiple techniques proved to be an informative survey approach, and the LH-PCR proved to be an effective technique applicable to the eukaryotic microbial community