Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation

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Title: Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation
Author(s): Crocetti, Gregory R.; Hugenholtz, Philip; Bond, Philip L.; Schuler, Andrew; Keller, Juerg; Jenkins, David; Blackall, Linda L.
Abstract: Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphate-accumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1 percent P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, > 80 percent of the A sludge bacteria were β-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the β-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing ≥ 98 percent sequence identity) related to Rhodocyclus spp. (94 to 97 percent identity) and Propionibacter pelophilus (95 to 96 percent identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the β-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR sludges are bacteria closely related to Rhodocyclus and Propionibacter.
Publication type: Journal article
Source: Applied and Environmental Microbiology, Vol. 66, no. 3 (Mar 2000), pp. 1175-1182
Publication year: 2000
Keyword(s): Actinobacteria; Activated sludge; Betaproteobacteria; Bioreactors; Fluorescence in situ hybridization; Oligonucleotide probes; Phosphorus; Phylogeny; Propionivibrio; Proteobacteria; Rhodocyclus; Ribosomal DNA; Sewage; Waste disposal
Publisher: American Society for Microbiology
ISSN: 0099-2240
Publisher URL: http://dx.doi.org/10.1128/aem.66.3.1175-1182.2000
Peer reviewed