Use of real-time PCR for group-specific quantification of aceticlastic methanogens in anaerobic processes: population dynamics and community structures.

The TaqMan quantitative PCR (QPCR) method was used to detect and quantify the 16S rRNA genes of aceticlastic methanogens at different taxonomic levels. Three different sets of primers coupled with a TaqMan probe for QPCR assays to detect the 16S rRNA genes of the order Methanosarcinales, as well as the families Methanosarcinaceae and Methanosaetaceae, were separately used. Using these primer and probe sets, the 16S rRNA genes of aceticlastic methanogens in samples from various anaerobic processes (i.e., nine pure cultures, batch experiment, and three different continuous processes including a full-scale digester), were monitored and quantified by QPCR assays. A batch experiment cultivating a mixture of aceticlastic methanogens, was conducted to monitor their population dynamics. Using this group-specific quantification method, the dynamics of a competition between two aceticlastic populations, as modulated by the acetate concentration, could well be described. The target 16S rRNA genes in environmental samples, collected from three different anaerobic processes treating sludge, cheese whey, and synthetic wastewaters, were additionally quantified. The quantified 16S rRNA gene concentrations for all samples successfully represented the community structures of the target methanogens, which were correlated accurately with the operational parameters of the anaerobic processes. It was also successful to demonstrate probe nesting of aceticlastic methanogens at the levels of order and family.