Live-Dead discrimination analysis is essential for molecular biology evaluation of advanced elimination processes of opportunistic pathogens at wastewater treatment plants

In respect to direct and indirect water reuse, the microbiological quality of treated wastewater is highly important. Semi-industrial ozone facilities were installed and applied 1.0 g ozone per g dissolved organic carbon (DOC) to the treated wastewater of the municipal wastewater treatment plant (WWTP). Wastewater samples directly before and after the ozone application were compared. Before DNA extraction all wastewater samples were treated with Propidium monoazide (PMA) to discriminate between viable and total bacteria. E. coli, enterococci, and Pseudomonas aeruginosa were quantified by polymerase chain reaction (qPCR) and the whole wastewater population was analyzed by metagenomic sequencing. It became obvious that live-dead discriminating methods are necessary to get reliable information about elimination efficiencies of ozone treatment. Comparing PMA-qPCR based results targeting taxon-specific gene markers with conventional cultivation data the presence of viable but not culturable (VBNC) cells for some indicator bacteria after ozone treatment became obvious. The PMA-qPCR experiments showed that the abundances of P. aeruginosa didn't change by ozone treatment, whereas E. coli and enterococci were much stronger reduced by ozonation. Illumina HiSeq sequencing results demonstrated high population similarities in water samples originating from ozone inflow sampling sides. Upon using PMA treatment after ozonation, population shifts became visible and underline the importance of PMA treatment for the evaluation of elimination and selection processes at WWTPs. Amongst a number of 14 most abundant genera identified in the inflow samples, 9 genera were found to be reduced, whereas 4 genera increased in relative abundance and 1 genus almost remained constant. The strongest increase in relative abundance after ozonation was detected for Oscillatoria spp., Microcoleus spp. and Nitrospira spp.. Hence, a continuous release of Pseudomonas spp. (including P. aeruginosa) to the downstream receiving body was confirmed. Regrowth experiments demonstrated a high prevalence of P. aeruginosa as part of the surviving bacterial population.