Revealing the Viable Microbial Community of Biofilm in a Sewage Treatment System Using Propidium Monoazide Combined with Real-Time PCR and Metagenomics

Microbial community composition, function, and viability are important for biofilm-based sewage treatment technologies. Most studies of microbial communities mainly rely on the total deoxyribonucleic acid (DNA) extracted from the biofilm. However, nucleotide materials released from dead microorganisms may interfere with the analysis of viable microorganisms and their metabolic potential. In this study, we developed a protocol to assess viability as well as viable community composition and function in biofilm in a sewage treatment system using propidium monoazide (PMA) coupled with real-time quantitative polymerase chain reaction (qPCR) and metagenomic technology. The optimal removal of PMA from non-viable cells was achieved by a PMA concentration of 4 μM, incubation in darkness for 5 min, and exposure for 5 min. Simultaneously, the detection limit can reach a viable bacteria proportion of 1%, within the detection concentration range of 10