Technical and biological study of aerobicgranular systems for hospital wastewatertreatment

The discharges of low-quality hospital and urban wastewater (WW) effluents into receiving aquatic ecosystems cause ecological damage and human health problems by spreading high loads of nutrients, pathogens and toxic compounds. The current Urban Wastewater Treatment Directive (91/271/CEE, European Commission, 1991) regulates the limit values for the emission of organic matter, nitrogen and phosphorus from municipal wastewater treatment plants (WWTPs). Although conventional activated sludge (CAS) systems can often reduce the concentration of these pollutants to meet the required limits, these systems have several drawbacks, including high energy consumption, excess sludge production, significant greenhouse gas emissions, low quality of the treated effluents, and design efficiency challenges, especially when dealing with low strength wastewater or fluctuating influent rates. In addition, these systems are not specifically designed to target emerging contaminants such as pharmaceutical active compounds (PhACs), personal care products, and endocrine-disrupting chemicals. As a result, they often fail to effectively remove these contaminants. Despite the current lack of regulations setting concentration limits for antibiotics in WW treated effluents, the removal of these compounds in CAS-based WWTPs co-treating hospital and urban WW is often scarce, creating a significant hotspot for the spread and proliferation of antibioticresistant bacteria (ARB) and antibiotic resistance genes (ARGs), and contributing to the worldwide emergence of antibiotic resistance. Therefore, the search for alternative bioprocesses to overcome the drawbacks of CAS needs to be encouraged. Aerobic granular sludge (AGS) operated in sequencing batch reactors (SBR) has a high potential for the improved treatment of urban and industrial WW and is currently considered the most promising technological alternative to overcome the disadvantages of CAS. Typical granular biomass allows the coexistence of different niches, enabling the metabolic versatility of the microbial communities and achieving excellent pollutant removal rates. The robustness of AGS for the treatment of complex WW containing recalcitrant and toxic compounds, such as antibiotics. Although several studies have investigated the feasibility of AGS for the efficient removal of PhACs in synthetic waters, to our knowledge, the evaluation of this technology for the treatment of real hospital WW has not been yet addressed. Thus, Chapter I focuses on the performance of AGS for the steady treatment of raw hospital effluents as a feasible approach to improve hospital WW management technologies. The characteristics of the granular biomass were not significantly affected, and the main microbial communities (Bacteria, Archaea and Fungi) were progressively acclimated to the healthcare WW inlet over time. The removal of several common PhACs was additionally analysed according to their correlation with the study area or ecological risks. Good removal performances of antibiotics, antiepileptics, and antidepressants drugs were achieved. In order to address the capacity of AGS to efficiently treat real hospital WW generating safe effluents, it is essential to determine the accumulation of ARGs and host ARB within the granular biomass. Therefore, Chapter II describes the development of an accurate methodology for the quantification of ARGs through sensitive and robust qPCR assays, supported by good primer design and validated protocols. The most relevant ARGs providing resistance to commonly prescribed antibiotic compounds were selected for their monitoring. The new molecular tools here designed were validated in samples retrieved from both natural and engineered environments, and the results highlighted a greater coverage of the new primer sets including a wider biodiversity of the bacterial populations carrying the different ARGs, plus a better design than the currently available molecular tools. Hence, after the validation of the new qPCR protocols, Chapter III provides an analysis of the occurrence and quantification of nine antibiotic compounds present in hospital WW and eleven ARGs within the AGS biomasses. Despite the excellent antibiotic removal performance achieved for the system, significantly high prevalence and enrichment of the targeted ARGs in the granular biomass were observed over time. Finally, Chapter IV presents the profiles of ARGs of five WWTPs operated at low temperatures as a reservoir of psychrophilic ARB, highlighting the usefulness of the new qPCR assays to monitor the occurrence and fate of antibiotic resistance markers and evaluate the role of conventional bioprocesses as a hotspots of ARGs. This study is particularly important for several reasons: first, cold temperature environments are the most widespread extreme habitats of the Earth's biosphere with a resistome that remains largely unknown; second, the role of WWTPs in the cryosphere as hotspots for the dissemination of antibiotic resistance has not been well established yet, and finally, the contribution of urban WW influents to the abundance and prevalence of ARGs in activated sludge samples needs to be thoroughly explored under these extreme temperature conditions. The overall results suggest that ARGs are ubiquitous and highly abundant in the cryosphere, despite the selection of microorganisms by low temperatures in WWTPs and natural cold environments. This study uses the recently developed quantitative methodologies for their application in environmental studies and strongly reinforces the global concern about the spread of antimicrobial resistance issues.