Flow cytometric characterization of bacterial abundance and physiological status in a nitrifying-denitrifying...

Flow cytometry has recently been presented as a research tool in the assessment of the viability/activity of activated sludge from municipal wastewater treatment plants, but it has not put in practice for industrial biotreatments yet. In this study, for the first time ever, the reliability and significance of the multiparameter flow cytometry applied to the biological nitrification-denitrification treatment of leachate have been evaluated. Using a double staining procedure (cFDA/PI), the viable, damaged, and dead subpopulations were determined, and the results were compared to those obtained with conventional methods, such as nitrogen and oxygen uptake rates or plate counting. Flow cytometry showed that viable cells represented approximately 47% of the total population, whereas active cells accounted for 90%. For both sludge from nitrification and denitrification processes, with less than 1% of them being also culturable in plate. Either flow cytometry or uptake rates revealed that health status of sludge remained constant throughout the biotreatment, which is consistent with the high recirculation rates. Under anaerobic starvation conditions, physiological status of sludge remained constant as well as specific oxygen and denitrification rates. Nevertheless, both the culturability in plate and the nitrification rate significantly decreased. These findings proved that multiparameter flow cytometry is a useful tool for the assessment of the viability and activity of sludge from a nitrification-denitrification biotreatment process. These results gathered all the bacterial communities in the sludge, so the decay in minority populations, such as nitrifying bacteria, requires the use of a complementary technique to evaluate specific activities.