Development of the Quantitative PCR Method for Candidatus ‘Accumulibacter phosphatis’ and Its Application to Activated Sludge

Abstract

To quantify Candidatus 'Accumulibacter phosphatis' in activated sludge, quantitative PCR method was developed utilizing SYBR GREEN I and a specific primer set targeted on the 16S rRNA gene of Candidatus 'Accumulibacter phosphatis'. Following optimization of PCR condition, specificity was evaluated based on the melting curve and the sequencing analysis of the PCR products with DNA extracted from activated sludge. Both the melting curve and the sequencing analysis of the PCR product showed that only the target DNA from Candidatus 'Accumulibacter phosphatis' was amplified. Standard curves with a series of tenfold dilution of the DNA from 16S rRNA gene fragment of Candidatus 'Accumulibacter phosphatis' gave R 2 values greater than 0.999. The minimum detection limit was 1.0×10 3 copies per reaction. The amount of Candidatus 'Accumulibacter phosphatis' in laboratory-scale and full-scale activated sludge samples were quantified both by the quantitative PCR method and by the FISH method. The quantification results by these two methods agreed satisfactorily, with an R 2 value of 0.6871 showing a statistically significant correlation (p<0.001). Thus, we developed a rapid quantification method by using quantitative PCR for the quantification of Candidatus 'Accumulibacter phosphatis' in activated sludge. INTRODUCTION Candidatus 'Accumulibacter phosphatis' is currently thought to be the most relevant polyphosphate accumulating organisms (PAOs) in the enhanced biological phosphorus removal (EBPR) process (Seviour et al., 2003; Mino et al., 1998). Hesselmann et al. (1999) and Crocetti et al. (2000) revealed that the bacteria closely related to Rhodocyclus (a member of the β-Proteobacteria) were progressively enriched and responsible for phosphorus removal in laboratory-scale EBPR reactors fed with acetate by using newly designed oligonucleotide probes. Hesselmann et al. (1999) reported that the bacteria was a coccobacillus and tentatively proposed its name as Candidatus 'Accumulibacter phosphatis'. In further study, Candidatus 'Accumulibacter phosphatis' has frequently been found to dominate many laboratory-scale EBPR cultures (Onuki et al., 2002; Liu et al., 2001; Oehmen et al., 2005) and has also been observed in abundance in full-scale wastewater treatment plants by using fluorescence in situ hybridization (FISH) (Wong et al., 2005; Beer et al., 2006).