Live-dead discrimination analysis, qPCR assessment for opportunistic pathogens, and population analysis at ozone wastewater treatment plants

In respect to direct and indirect water reuse, the microbiological quality of treated wastewater is highly important. Conventional wastewater treatment plants are normally not equipped with advanced technologies for the elimination of bacteria. Molecular biology analyses were combined with live-dead discrimination analysis of wastewater population using Propidium monoazide (PMA) to study population shifts during ozonation (1 g ozone/g DOC) at a municipal wastewater treatment plant. Escherichia coli, enterococci, and Pseudomonas aeruginosa were quantified by polymerase chain reaction (qPCR) and the whole wastewater population was analyzed by metagenomic sequencing. The PMA-qPCR experiments showed that the abundances of P. aeruginosa didn't change by ozone treatment, whereas a reduction was observed for E. coli and enterococci. Results comparing conventional cultivation experiments with PMA-qPCR underlined the presence of viable but not culturable cells (VBNC) and their regrowth potential after ozone treatment. Illumina HiSeq sequencing results with and without PMA treatment demonstrated high population similarities in water samples originating from ozone inflow sampling sides. Upon using PMA treatment after ozonation, population shifts became visible and also underlined the importance of PMA treatment for the evaluation of elimination and selection processes during ozonation 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. Beside this, 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. Summing up, molecular biology analyses in combination with live-dead discrimination are comprehensive methods to evaluate the elimination processes targeting specific species and/or whole microbial populations.