Abstract
While the research into the activated sludge (AS) microbiome using next-generation sequencing (NGS) are increasingly being published, they still apply to a few wastewater treatment plants (WWTPs). Little information can be found in the scientific literature on the microbial composition of treated wastewater (TW) and bacterial activity in real WWTPs facilities. In this study, AS and TW are compared in terms of their physico-chemical parameters and bacterial community composition, with particular emphasis on nitrifying bacteria, based on the results from 16S rRNA amplicon sequencing using DNA and complementary DNA (cDNA) data and fluorescence in situ hybridization (FISH). Using a 24-h composite sample of TW rather than a grab sample yields highly representative microbial community profiles. The research revealed a decrease in biodiversity in months with lower sludge temperatures both in AS and TW. Differences among groups of nitrifying bacteria were observed during the monitoring of WWTP, suggesting that their presence in TW may not be a random occurrence but could serve as an indicator of physicochemical and operational changes within biological reactors. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-025-34503-4.