Combination of flow cytometry and molecular analysis to monitor the effect of UVC/H 2 O 2 vs UVC/H 2 O 2 /Cu-IDS processes on pathogens and antibiotic resistant genes in secondary wastewater effluents

The efficiency of a new Advanced Oxidation Process (AOP), namely the photo Fenton like process UV- C/H 2 O 2 /IDS-Cu, in removing determinants of antibiotic resistance and pathogenic bacteria was compared to a consolidated AOP (namely UV-C/H 2 O 2 ) in a secondary treated municipal WasteWater (WW). A re- ductionist experimental laboratory-based approach was applied on real WW and the parameters were collected by an alternative integrated approach using (i) flow cytometry to enumerate bacteria and test for the fitness of the bacterial communities and (ii) molecular analyses to define the community com- position (16S rRNA amplicon sequencing) and the abundances of Antibiotic Resistance Genes (ARGs) and of the class 1 integron ( int I1 gene) (by quantitative PCR). The same approach was applied also to post- treatment regrowth tests (24 h) to define the potential persistence of the tested parameters. These exper- iments were performed in both, human pathogens favorable conditions (HPC, in rich medium and 37 °C) and in environmental mimicking conditions (EMC, original WW and 20 °C). UV-C/H 2 O 2 /IDS-Cu process re- sulted to be more effective than the UV-C/H 2 O 2 in inactivating bacterial cells in the EMC post-treatment regrowth experiments. Both AOPs were efficiently abating potential human pathogenic bacteria and ARGs in the HPC regrowth experiments, although this trend could not be detected in the measurements taken immediately after the disinfection. In comparison with the UV-C/H 2 O 2 , the UV-C/H 2 O 2 /IDS-Cu process did not apparently offer significant improvements in the abatement of the tested parameters in the WW effluent but, by evaluating the results of the regrowth experiments it was possible to extrapolate more complex trends, suggesting contrasting efficiencies visible only after a few hours. This study offers a de- tailed view on the abatement efficiency of microbiological/genetic parameters for the UV-C/H 2 O 2 /IDS-Cu process, calling for technical adjustments for this very promising technology. At the same time, our re- sults clearly demonstrated the inadequacy of currently applied methodologies in the evaluation of specific parameters (e.g. determinants of antibiotic resistance and pathogenic bacteria) in WW.