The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is shed in the feces of infected people. As a consequence, genomic RNA of the virus can be detected in wastewater. Although the presence of viral RNA does not inform on the infectivity of the virus, this presence of genetic material raised the question of the effectiveness of treatment processes in reducing the virus in wastewater and sludge. In this work, treatment lines of 16 wastewater treatment plants were monitored to evaluate the removal of SARS-CoV-2 RNA in raw, processed waters and sludge, from March to May 2020. Viral RNA copies were enumerated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in 5 different laboratories. These laboratories participated in proficiency testing scheme and their results demonstrated the reliability and comparability of the results obtained for each one. SARS-CoV-2 RNA was found in 50.5% of the 101 influent wastewater samples characterized. Positive results were detected more frequently in those regions with a COVID-19 incidence higher than 100 cases per 100,000 inhabitants. Wastewater treatment plants (WWTPs) significantly reduced the occurrence of virus RNA along the water treatment lines. Secondary treatment effluents showed an occurrence of SARS-CoV-2 RNA in 23.3% of the samples and no positive results were found after MBR and chlorination. Non-treated sludge (from primary and secondary treatments) presented a higher occurrence of SARS-CoV-2 RNA than the corresponding water samples, demonstrating the affinity of virus particles for solids. Furthermore, SARS-CoV-2 RNA was detected in treated sludge after thickening and anaerobic digestion, whereas viral RNA was completely eliminated from sludge only when thermal hydrolysis was applied. Finally, co-analysis of SARS-CoV-2 and F-specific RNA bacteriophages was done in the same water and sludge samples in order to investigate the potential use of these bacteriophages as indicators of SARS-CoV-2 fate and reduction along the wastewater treatment.

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Monitoring bacterial communities in a drinking water treatment plant (DWTP) may help to understand their regular operations. Bacterial community dynamics in an advanced full-scale DWTP were analyzed by 16S rRNA metabarcoding, and microbial water quality indicators were determined at nine different stages of potabilization: river water and groundwater intake, decantation, sand filtration, ozonization, carbon filtration, reverse osmosis, mixing chamber and post-chlorination drinking water. The microbial content of large water volumes (up to 1100 L) was concentrated by hollow fiber ultrafiltration. Around 10 million reads were obtained and grouped into 10,039 amplicon sequence variants. Metabarcoding analysis showed high bacterial diversity at all treatment stages and above all in groundwater intake, followed by carbon filtration and mixing chamber samples. Shifts in bacterial communities occurred downstream of ozonization, carbon filtration, and, more drastically, chlorination. Proteobacteria and Bacteroidota predominated in river water and throughout the process, but in the final drinking water, the strong selective pressure of chlorination reduced diversity and was clearly dominated by Cyanobacteria. Significant seasonal variation in species distribution was observed in decantation and carbon filtration samples. Some amplicon sequence variants related to potentially pathogenic genera were found in the DWTP. However, they were either not detected in the final water or in very low abundance (<2%), and all EU Directive quality standards were fully met. A combination of culture and high-throughput sequencing techniques may help DWTP managers to detect shifts in microbiome, allowing for a more in-depth assessment of operational performance.

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