Molecular methods for the analysis of microbial communities: application to natural environments affected by anthropogenic modifications.

Microbes are ubiquitous in the environment and are key component of Earth Biota, covering fundamental roles for the functioning of ecosystems. A comprehensive characterization of such astonishing diversity is of fundamental importance to better understand the functioning of ecosystems, and constitutes a starting point for new technological applications and for issues of natural conservation. Our understanding of the microbial world, however, has been heavily limited by the impossibility to apply classical microbiological techniques to natural populations, mostly due to our incapacity of growing them in laboratory media. In this regard, a great advance came from molecular biology and to its application to the study of molecular markers (primarily SSU rRNAs). Moreover, with the drop in costs of New Generation Sequencing (NGS) technologies, and with the consequent increase of their application to microbial ecology studies, a substantial boost in our understanding of natural microbial communities has been achieved. Molecular methods are actually the most comprehensive alternative for the study of natural microbial communities; and my PhD work has been primarily focused on the application of such methodologies to the study of microbial communities in natural environments affected by anthropogenic modifications. In this thesis, principles of the molecular analysis of microbial communities were covered, presenting the results of two case studies. Two molecular methodologies were applied: terminal restriction fragments length polymorphism (T- RFLP) and next generation sequencing (NGS) with MiSeq platform. In one case (chapter 4, 6, and 7), activities connected to the European project “DEMETRA” were presented. General aim of the project was to provide innovative instruments for the monitoring of the risk for biodiversity connected to the (hypothetical) introduction of genetically modified (GM) crops in the environment of the regional park of Migliarino San Rossore Massacciuccoli. For this reason, a primary step was the characterization of biodiversity at various levels, including those of soil microbial communities. Subject of the study were bacterial and fungal populations in soils from. Waiting for the whole botanical/forestry, entomological and microbiological data set will be processed to determine a risk index of GM crops, we have utilized the microbiological data to evaluate the long-lasting effects of a change within a single land-use category, by comparing microbial diversity in soils from two natural forests and a forest converted to poplar plantation about thirty years ago within the Park. Results from T-RFLP and NGS approach showed high concordance. Both identified the change in land-use as a major perturbative factor for bacterial 1communities (and also for fungal ones, considering T-RFLP results), individuating a decrease in richness in microbial communities from soils of the converted poplar plantation. Land-use was a stronger factor respect to vegetation cover in shaping microbial community. In fact, a higher similarity in community composition was found between the two natural forests (distant from each other in space, and characterized by a different vegetation cover), than between the converted and the natural poplar plantation (really close in space and characterized by the same vegetation cover) In the second case (chapter 5, 7, and 9), activities connected to the European project “MAPMED” were presented. General aim of the project was to improve the environmental sustainability of tourist coastal areas in the Countries of the Mediterranean Sea Basin and to optimize, validate, and transfer tools for the sustainable management of tourist ports with regard to monitoring and reduction of marine pollution. For this reason, the diversity and structure of bacterial and archaeal communities in sediments from three ports in the Mediterranean Sea (Cagliari, Italy; El Kantaoui, Tunisia; Heraklion Greece) were characterized. Results indicate that microbial communities in sediments from the three ports were different and that port of origin constituted the main grouping factor. The overall diversity levels however, regardless of the geographic position of port, where low, indicating that the port basin represent a peculiar habitat unconnected with well-established north-south and east- west gradients of diversity and environmental conditions at the level of Mediterranean basin. A peculiar community composition was observed in sediment from a shipyard in the port of Heraklion, highly different from the rest of samples. Activities linked to shipyards are characterized by pollutants and impacts strongly different from the rest of the touristic port environment, ultimately affecting microbial communities in sediments. The use of T-RFLP on three different molecular markers (16S rRNA on bacterial communities, 16S rRNA on archaeal communities, and dsrAB) as methodology of choice for monitoring plans was evaluated and compared to results from NGS analysis to provide a mean of validation. T-RFLP was able to recapture main results from NGS fairly well, and the marker that performed better was dsrAB. For those reasons, T-RFLP could be effectively be used as the routine technique in the monitoring of marine pollution in sediments of touristic ports from the Mediterranean Sea Basin, although its effectiveness should be exceptionally corroborated by NGS analysis.