Evaluation of concentration and extraction workflows for enhanced bacterial 16S rRNA profiling of urban wastewater samples

This study investigated the effects of various sample concentration and extraction workflows on the alpha and beta diversities of bacterial communities in influent wastewater samples from three wastewater treatment plants (WWTPs). The workflows that incorporated DNeasy Blood & Tissue (DBT) Kit consistently demonstrated the highest alpha diversity metrics (% total observed in study), indicating superior efficacy in capturing microbial diversity. In contrast, workflows that included the DNeasy PowerSoil Pro (DPP) Kit exhibited the lowest alpha diversity (% total), likely due to suboptimal lysis efficiency. Beta-diversity analysis revealed significant differences in microbial community composition of wastewater samples across the WWTPs, driven by workflows and temporal variations. Notably, Proteobacteria was the dominant phylum, while variations in the abundance of Firmicutes and Actinobacteria were also observed, underscoring the methodological influence on community profiling. The study also revealed significant variations in the Firmicutes/Bacteroidetes (F/B) ratio across workflows and WWTP samples, suggesting potential correlations with local health demographics. Our findings emphasize the critical role that sample concentration and extraction workflows play in bacterial community analyses, and that high variability of results between workflows may often prevent meaningful data comparison. Quantifying and reporting metric for the level of bacterial 16S rRNA capture together with details of the sample concentration and extraction protocols will improve comparison of diverse bacterial populations in wastewater environments. This research advances the understanding of bacterial dynamics in WWTP influent samples, enhancing wastewater surveillance efforts.