title: Use of real-time PCR with propidium monoazide for enumeration of viable Escherichia coli in anaerobic digestion.
abstract: A combination of propidium monoazide (PMA) with real-time quantitative polymerase chain reaction (PMA-qPCR) was optimized to enumerate only viable Escherichia coli in anaerobic digestion processes. Repeating the PMA treatment twice and a final concentration of 100 μM resulted in an effective exclusion of DNA from heat-treated E. coli cells. In three anaerobic digestion processes, real-time PCR, PMA-qPCR, and the most probable number method (MPN) were used to estimate the numbers of total, viable, and culturable E. coli cells, respectively. Culturable concentrations of fecal coliforms were also measured by the membrane filter method. For thermophilic digestion, the reductions in total and viable E. coli cells from the digester influent to the effluent were significantly lower than those in culturable cells and fecal coliforms by two to four orders of magnitude. For mesophilic digestion, the differences in the reductions in E. coli and fecal coliforms counts were less than two orders of magnitude. Based on the measurements of viable E. coli determined by the PMA-qPCR method, the microbial quality of digester effluents was discussed for agricultural application, and pasteurization after anaerobic digestion was suggested for the destruction of viable pathogens.
authors.affiliation: Department of Civil and Environmental Engineering, Tohoku University, 6-6-06 Aoba, Aramaki, Aobaku, Sendai 980-8579 Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Ruike
authors.affiliation: Department of Civil and Environmental Engineering, Tohoku University, 6-6-06 Aoba, Aramaki, Aobaku, Sendai 980-8579 Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Higashimori
authors.affiliation: Department of Civil and Environmental Engineering, National Institute of Technology, Hachinohe College, 16-1 Uwanotai, Tamonoki, Hachinohe 039-1192, Japan E-mail: yaguchi-z@hachinohe-ct.ac.jp.
authors.forename: Junichi
authors.initials: J
authors.lastname: Yaguchi
authors.affiliation: Department of Civil and Environmental Engineering, Tohoku University, 6-6-06 Aoba, Aramaki, Aobaku, Sendai 980-8579 Japan.
authors.forename: Yu-You
authors.initials: YY
authors.lastname: Li
mesh_terms: D000383:Agriculture; D001386:Azides; D004926:Escherichia coli / Q000235:genetics* / Q000302:isolation & purification; D050296:Microbial Viability; D011419:Propidium / Q000031:analogs & derivatives*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D014871:Water Microbiology
chemical_list: D001386:Azides; C533957:propidium monoazide; D011419:Propidium