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 102-108 CFU/mL (colony forming unit/mL), showing its effectiveness in removing interference from dead cells. Under the optimal conditions, the result of PMA-metagenomic sequencing revealed that 6.72% to 8.18% of non-viable microorganisms were influenced and the composition and relative abundance of the dominant genera were changed. Overall, this study established a fast, sensitive, and highly specific biofilm viability detection method, which could provide technical support for accurately deciphering the structural composition and function of viable microbial communities in sewage treatment biofilms.

Response of soil microbial community structure and function to the sewage leakage: A case study of a 25-year-old cesspool.

Providing many millions of rural households with decentralized sanitation facilities remains challenging. In undeveloped areas, cesspools have still been widely used due to technologically simple and low-cost. However, the influence of cesspools on the surrounding soil remains unclear. In this study, we investigated the influence of a 25-year-old household cesspool on soil physicochemical factors, microbial community composition and function, pathogens and antibiotic resistance genes (ARGs). Soil at the depth around the sewage liquid level (D70) was mostly disturbed where TOC, NO3-N and TP was increased to 16.8 g/kg, 18.2 mg/kg and 1.02 mg/kg respectively. Correspondingly, the element cycling genes of carbon fixation, methanotrophy, nitrogen fixation, ammonia oxidation, and nitrate reduction etc., were increased at D70. Notably, human derived pathogens such as Enterobacter, Salmonella, Pseudomonas aeruginosa, Klebsiella pneumoniae, Prevotella, and Vibrio were highly enriched by 5-10 folders in D70, indicating the potential health risk to human. Mantel tests suggested that EC, TP, pH, NH3-N and particularly NO3-N are important factors that influence the microbial community and element cycling genes in cesspool-affected soil. Overall, this study revealed the impact of household cesspool leakage on the surrounding soil and provided information for the selection and construction of basic sanitation facilities in poor regions.