Petroleum hydrocarbon contamination (PHC) is an issue of major concern worldwide. These compounds represent the most common environmental pollutants and their cleaning up is mandatory. The main goal of this research was to analyze microbial communities in a site in southern Italy characterized by the presence of hydrocarbons of natural origin by using a multidisciplinary approach based on microbiological, geological and hydrological investigations. Bacterial communities of two springs, the surrounding soils, and groundwater were studied through a combination of molecular and culture-dependent methodologies to explore the biodiversity at the study site, to isolate microorganisms with degradative abilities, and to assess their potential to develop effective strategies to restore the environmental quality. Next-generation sequencing revealed the dominance of species of the Proteobacteria phylum but also the presence of other autochthonous hydrocarbon-oxidizing microorganisms affiliated to other phyla (e.g., species of the genera Flavobacterium and Gordonia). The traditional cultivation-based approach led to the isolation and identification of 11 aerobic hydrocarbon-oxidizing proteobacteria, some of which were able to grow with phenanthrene as the sole carbon source. Seven out of the 11 isolated bacterial strains produced emulsion with diesel fuel (most of them showing emulsifying capacity values greater than 50%) with a high stability after 24 h and, in some cases, after 48 h. These results pave the way for further investigations finalized at (1) exploiting both the degradation ability of the bacterial isolates and/or microbial consortia to remediate hydrocarbon-contaminated sites and (2) the capability to produce molecules with a promoting effect for oil polluted matrices restoration.

Knowledge about the processes governing subsurface microbial dynamics in and to groundwater represents an important tool for the development of robust, evidence-based policies and strategies to assess the potential impact of contamination sources and for the implementation of appropriate land use and management practices. In this research, we assessed the effectiveness of using microorganisms as natural tracers to analyze subsurface dynamics in a low-permeability system of northern Italy. Microbial communities were investigated through next-generation sequencing of 16S rRNA gene both to study hydraulic interconnections in clayey media and to verify the efficacy of outcropping clayey horizons in protecting groundwater against contamination. During the observation period, a rapid water percolation from the ground surface to the saturated medium was observed, and the mixing between lower-salinity fresh-infiltration waters and higher-salinity groundwater determined the formation of a halocline. This rapid percolation was a driver for the transport of microorganisms from the topsoil to the subsurface, as demonstrated by the presence of soil and rhizosphere bacteria in groundwater. Some of the species detected can carry out important processes such as denitrification or nitrate-reduction, whereas some others are known human pathogens (Legionella pneumophila and Legionella feeleii). These findings could be of utmost importance when studying the evolution of nitrate contamination over space and time in those areas where agricultural, industrial, and civil activities have significantly increased the levels of reactive nitrogen (N) in water bodies but, at the same time, could highlight that groundwater vulnerability of confined or semi-confined aquifers against contamination (both chemical and microbiological) could be higher than expected.

The studies upstream of the petroleum industry include oil and gas geological exploration and are usually focused on geological, structural, geophysical, and modeling techniques. In this research, the application of a coupled microbiological–isotopic approach was explored to assess its potential as an adequate characterization and monitoring tool of geofluids in oilfield areas, in order to expand and refine the information acquired through more consolidated practices. The test site was selected within the Val d’Agri oilfield, where some natural hydrocarbon springs have been documented since the 19th century in the Tramutola area. Close to these springs, several tens of exploration and production wells were drilled in the first half of the 20th century. The results demonstrated the effectiveness of the proposed approach for the analysis of fluid dynamics in complex systems, such as oilfield areas, and highlighted the capacity of microbial communities to “behave” as “bio-thermometers”, that is, as indicators of the different temperatures in various subsurface compartments.

Knowledge about the hydrogeological behaviour of heterogeneous low-permeability media is an important tool when designing anthropogenic works (e.g., landfills) that could potentially have negative impacts on the environment and on people’s health. The knowledge about the biogeochemical processes in these media could prevent “false positives” when studying groundwater quality and possible contamination caused by anthropogenic activities. In this research, we firstly refined knowledge about the groundwater flow field at a representative site where the groundwater flows within an evaporite-bearing low-permeability succession. Hydraulic measurements and tritium analyses demonstrated the coexistence of relatively brief to very prolonged groundwater pathways. The groundwater is recharged by local precipitation, as demonstrated by stable isotopes investigations. However, relatively deep groundwater is clearly linked to very high tritium content rainwater precipitated during the 1950s and 1960s. The deuterium content of some groundwater samples showed unusual values, explained by the interactions between the groundwater and certain gases (H₂S and CH₄), the presences of which are linked to sulfate-reducing bacteria and methanogenic archaea detected within the saturated medium through biomolecular investigations in the shallow organic reach clayey deposits. In a wider, methodological context, the present study demonstrates that interdisciplinary approaches provide better knowledge about the behaviour of heterogeneous low-permeability media and the meaning of each data type.

The increase in oil production from petroleum reservoirs has led to studies examining the effects of these activities on groundwater quality. Oily wastewater associated with oil production is often reinjected through abandoned wells into the unproductive portions of the reservoir to avoid its discharge on the surface. The reinjection process is designed to be environmentally friendly and to exclude direct interactions between injected fluids and the surrounding groundwater; nevertheless, the evaluation of the compatibility between this process and the protection of the surrounding environment is of utmost importance when oilfields are located within sensitive and protected areas. The present work aimed to evaluate the impact of the oily wastewater reinjection into a long-term and high-rate disposal well in the Val d’Agri oilfield (Southern Italy). Previous preliminary investigations carried out at the study site led researchers to hypothesize the possible hydrocarbon contamination of the shallower aquifer caused by reinjection well integrity issues. Our strategy is based on an integrated and multidisciplinary approach involving isotopic (stable isotopes ²H and ¹⁸O), chemical, and microbiological (characterization of bacterial and archaeal communities) analyses. After a comprehensive and meticulous examination of the research data, it has been ascertained that significant discrepancies exist between the shallow and reinjection water systems. This allowed us to clarify the area’s complex flow dynamics and exclude hydrocarbon contamination of spring waters caused by the reinjection process.