Study of microbial strains and communities for their capability of biotransforming/biodegrading perfluoroalkyl substances (PFAS), to develop strategies for the biological remediation of water bodies and soils polluted by PFAS

Poly- and perfluoroalkyl substances (PFASs) are a class of highly fluorinated xenobiotic compounds with toxic and carcinogenic properties that make them significant pollutants with major implications for both human and environmental health. These molecules can travel far from their point of origin via water cycles and animal migration and exhibit a high degree of bioaccumulation and biomagnification, leading to increased concentrations along the food chain. Their half-life in the human body varies by compound but can span several years. Although their properties as persistent environmental pollutants and long-term toxicants have been known for decades, research is ongoing to develop effective methods for their removal and degradation from environmental matrices. Current chemical-physical remediation processes—most notably filtration followed by incineration—present economic, energetic, and environmental drawbacks. Conversely, literature has shown that certain microorganisms can mitigate PFAS pollution. Among these are strains of Pseudomonas sp., Labrys sp. F11, and Acidimicrobium sp. A6, which have demonstrated the ability to degrade, defluorinate, or accumulate these contaminants. The application of such microorganisms could support the development of ecologically and economically sustainable bioremediation strategies. The objective of this Ph.D. project was to study microbial communities present in environmental matrices contaminated with PFASs—such as soil, groundwater, and spent activated carbon—in order to obtain and characterize bacterial strains in terms of their tolerance to PFASs and, potentially, their capacity for biotransformation or bioaccumulation of these compounds. Specifically, we investigated the interaction between bacterial isolates and four representative PFAS molecules: 1H,1H,2H,2H-tridecafluoro-1-octanol (6:2 FTOH), 1H,1H,2H,2H-perfluoro-1-decanol (8:2 FTOH), tridecafluorohexane-1-sulfonic acid (PFHxS), and perfluorooctanoic acid (PFOA). The matrices used in this study were collected from an area in the province of Vicenza, Italy, which has been affected by PFAS contamination for 43 years due to industrial wastewater discharge. Measurements revealed that PFAS concentrations in surface and groundwater could exceed 1,000 ng/L, reaching peaks between 40,000 and 50,000 ng/L. Information regarding the levels of contamination in soils are generally lacking and rare, but recent observations by ARPAV have highlighted low concentrations of PFASs in the soils of the polluted region. PFOA resulted the only PFASs present at detectable concentrations, but never higher than 4 µg/kg dw (Microinquinanti Organici - ARPAV). Enrichment cultures were prepared using environmental samples from an high PFASs polluted area and a defined minimal medium supplemented with the two fluorotelomers and PFHxS at 200 mg/L, or PFOA at 600 mg/L. The aim was to isolate microbial strains able to withstand these concentrations and potentially biotransform the compounds. A total of 50 bacterial strains were isolated and identified by partial sequencing of the 16S rRNA gene, mainly belonging to the families Pseudomonadaceae and Alcaligenaceae. Their growth in the presence of PFASs and the effects of these compounds on microbial cells were evaluated using various approaches: i) growth assessment in liquid media under both proliferative (growing) and non-proliferative (resting) conditions; ii) transmission electron microscopy (TEM) analysis of exposed cells; iii) ζ-potential measurements on treated and untreated cells; iv) preliminary evaluation of biofilm-forming capabilities, with and without PFAS exposure, using Calgary Biofilm Devices (CBD) and confocal fluorescence microscopy. The selected bacterial strains were resistant to the PFAS compounds tested; under growing conditions, most did not show toxic effects up to 1,000 mg/L of PFHxS or PFOA. Some strains even showed increased turbidity (OD600) in liquid culture: Brucella sp. 8S10 and Ochrobactrum sp. 8S11 when exposed to PFHxS and PFOA; Labrys sp. C11 to fluorotelomers; and Bacillus sp. CO5 at 600 mg/L of PFOA. Under resting conditions, Labrys sp. C11 and Achromobacter sp. 8S3 showed minimal inhibition of cell viability when exposed to PFHxS. TEM observations confirmed the absence of morphological damage in these strains. The ζ-potential data aligned with the results of the growth and resting condition assays: strains like Labrys sp. C11 and Achromobacter sp. 8S3 showed only minimal changes upon PFAS exposure, further suggesting their resistance. Our investigation also indicated that these strains are capable of biofilm formation, as confirmed by CBD assays. It is hypothesized that PFAS compounds may affect biofilm development, slowing or in some cases inhibiting its formation. Thus, we investigated the effects of PFASs on bacterial strains isolated from four contaminated matrices with high PFAS concentrations, primarily belonging to the families Pseudomonadaceae and Alcaligenaceae. These microorganisms demonstrated high tolerance, with toxic effects generally observed only above 600 mg/L—a concentration rarely reached under natural conditions. Among the most promising strains are those of the genera Pseudomonas, Labrys, and Brucella. The specific mechanisms underlying their tolerance were not explored in this thesis but will be the subject of future research. During the research period abroad in Ireland, carried out in the Laboratory of Professor Piet Lens, in the University of Galway, it was evaluated the effects of per- and polyfluoroalkyl substances (PFASs) on an anaerobic granular sludge (AGS) system. AGS is a complex microbial consortium capable of carrying out multiple biochemical processes, including methanogenesis. Our focus was on its ability to generate methane when fed with acetate and how this metabolic activity might be impacted by the presence of various PFASs. This study aimed to assess the influence of four PFASs—6:2 FTOH, 8:2 FTOH, PFHxS, PFOA—on the AGS using acetate as the sole carbon source. The AGS used in this experiment originated from a treatment system processing milk and dairy by-product waste. While the two fluorotelomers caused marginal changes in methane production, PFHxS and PFOA greatly inhibited acetoclastic methanogenesis. Depending on the concentration, the AGS could recover its activity after a certain number of days when incubated with PFASs. The 50% Inhibitory concentration (IC50) of the methane yield was estimated to be 278.98 (± 3.2) mg/L for PFOA and 1,091.9 (± 26.78) mg/L for PFHxS. PFAS changed differently the archaeal and eubacterial populations. PFASs exposure induced an increase in the relative abundance of the genus Sulfurospirillum, among the eubacteria, in the samples treated with 2,000 mg/L of 8:2 FTOH and 600 mg/L of PFOA, which presence grew to represent 16.65% and 45.4% of all reads in those samples.