ABSTRACT
“
Candidatus
Accumulibacter” and total bacterial community dynamics were studied in two lab-scale enhanced biological phosphorus removal (EBPR) reactors by using a community fingerprint technique, automated ribosomal intergenic spacer analysis (ARISA). We first evaluated the quantitative capability of ARISA compared to quantitative real-time PCR (qPCR). ARISA and qPCR provided comparable relative quantification of the two dominant “
Ca
. Accumulibacter” clades (IA and IIA) detected in our reactors. The quantification of total “
Ca
. Accumulibacter” 16S rRNA genes relative to that from the total bacterial community was highly correlated, with ARISA systematically underestimating “
Ca
. Accumulibacter” abundance, probably due to the different normalization techniques applied. During 6 months of normal (undisturbed) operation, the distribution of the two clades within the total “
Ca
. Accumulibacter” population was quite stable in one reactor while comparatively dynamic in the other reactor. However, the variance in the clade distribution did not appear to affect reactor performance. Instead, good EBPR activity was positively associated with the abundance of total “
Ca
. Accumulibacter.” Therefore, we concluded that the different clades in the system provided functional redundancy. We disturbed the reactor operation by adding nitrate together with acetate feeding in the anaerobic phase to reach initial reactor concentrations of 10 mg/liter NO
3
-N for 35 days. The reactor performance deteriorated with a concomitant decrease in the total “
Ca
. Accumulibacter” population, suggesting that a population shift was the cause of performance upset after a long exposure to nitrate in the anaerobic phase.
ABSTRACT
We investigated the fine-scale population structure of the “
Candidatus
Accumulibacter” lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene (
ppk1
) as a genetic marker. We retrieved fragments of “
Candidatus
Accumulibacter” 16S rRNA and
ppk1
genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and
ppk1
were largely congruent, with
ppk1
granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the “
Candidatus
Accumulibacter” lineage. Sequences from at least five clades of “
Candidatus
Accumulibacter” were recovered by
ppk1
-targeted PCR, and subsequently, specific primer sets were designed to target the
ppk1
gene for each clade. Quantitative real-time PCR (qPCR) assays using “
Candidatus
Accumulibacter”-specific 16S rRNA and “
Candidatus
Accumulibacter” clade-specific
ppk1
primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total “
Candidatus
Accumulibacter” lineage and the relative distributions and abundances of the five “
Candidatus
Accumulibacter” clades. The qPCR-based estimation of the total “
Candidatus
Accumulibacter” fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using
ppk1
, demonstrating the power of
ppk1
as a genetic marker for detection of all currently defined “
Candidatus
Accumulibacter” clades. The relative distributions of “
Candidatus
Accumulibacter” clades varied among different EBPR systems and also temporally within a system. Our results suggest that the “
Candidatus
Accumulibacter” lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.