One health (Integrated approach to health focused on interactions between animals, humans and the environment with attention to inland valorization)

Landfill leachate represents one of the most complex environmental challenges due to its highly pollutant impact. In fact, it is rich in ammonium nitrogen (NH4+-N), recalcitrant organic compounds and heavy metals. Traditional wastewater treatment plants (WWTPs) struggle to handle this pollutant, especially when leachate matures, standing for a long time in landfill, and becomes increasingly difficult to be biologically treated. This research aims to explore innovative solutions to this pressing issue by combining microbial engineering techniques with advanced biofilm-based technologies. In the first phase, the present study analyzed the microbial composition of activated sludge derived from the Porto Sant’Elpidio, WWTP, located in central Italy. This plant annually collects and treats large volumes of landfill leachate from a large surrounding area. Using metagenomic sequencing of the 16S rRNA gene, native microbial communities were identified and evaluated for their ability to tolerate high concentrations of ammonium, exceeding 350 mg/L. Through a selection process, called Repetitive Re-Inoculum Assay (RRIA), microbial diversity was significantly reduced, allowing the enrichment of specialized and highly efficient nitrogen-removing species. The predominant bacterial families identified included Alcaligenaceae, Nitrosomonadaceae, Chitinophagaceae, and Comamonadaceae, which demonstrated ammonium removal rates of up to 85% in a leachate-based medium. Additionally, three genera – Klebsiella sp., Castellaniella sp., and Acinetobacter sp. were isolated and found capable of converting ammonium into gaseous nitrogen by combining heterotrophic nitrification and aerobic denitrification. These coupled processes minimize the release of harmful intermediates such as nitrites and nitrates, further enhancing the efficiency and sustainability of the treatment. The second phase of our study focused on applying these enriched microbial communities to develop a moving bed biofilm (MBB) system. This approach leverages the capacity of biofilms to concentrate active biomass and improve the efficiency of biological treatment processes. The biofilms were cultivated on high-density polyethylene (HDPE) carriers, and their development was monitored using advanced techniques such as Crystal Violet staining and confocal microscopy. The results were remarkable: the MBBs achieved an ammonium removal rate of up to 80% within just 24 hours, with a nitrification efficiency ten times higher than conventional activated sludge where bacteria were in planktonic phase. Metagenomic analyses confirmed the critical role of bacterial families such as Chitinophagaceae, Comamonadaceae, Sphingomonadaceae, and Nitrosomonadaceae in driving nitrification and denitrification processes. Moreover, MBBs exhibited exceptional robustness under varying environmental conditions, including low temperatures (as low as 10°C) and high salinity levels, making them a versatile solution for real-world applications. The results so far achieved are very promising in the attempt to overcome the limitations of traditional WWTPs in handling highly polluted leachates. In fact, targeted microbial community selection, followed by bacteria integration into mobile biofilm systems, has proven to be a highly efficient and sustainable solution for landfill leachate treatment. The initial phase of this study provided a strong theoretical foundation to identify and select key microorganisms from the sludge in order to obtain an optimized mixture with elevated efficiency in ammonia removal. In the second phase, we used these selected bacterial communities to generate biofilms demonstrating the practical potential of MBBs to significantly enhance the performance of existing facilities. Our study represents an advancement in wastewater management, paving the way for large-scale application of the modern technologies as MBBs and offering an innovative and effective response to global environmental challenges.