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

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% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were beta-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 beta-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing >/=98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) and Propionibacter pelophilus (95 to 96% 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 beta-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.

FIG. 1

Profiles of soluble extracellular phosphate-P (□), extracellular acetate (●), cellular PHA (○), and cellular carbohydrate (▴) during the anaerobic and aerobic reactor cycle stages of the P sludge of Bond et al. (8).

FIG. 2

Micrographs of mixed liquors from SBRs. (A and B) Bright-field micrographs of GRC sludge as operated according to data in Table 1. (A) Methylene blue stain. Standard-arrowed purple clusters of cells are those containing polyphosphate, while diamond-ended-arrowed blue cells do not contain polyphosphate. The bar is for both panels and is 6 μm. (B) Gram stain. Standard-arrowed orange clusters of cells match the morphology and size of the purple cells in panel A. Diamond-ended-arrowed pink cells match those of the blue cells in panel A. Morphologically identified “Nostocoida limicola” II can be seen as filaments of gram-positive and gram-negative cells. (C and D) Confocal laser scanning micrographs of sludges dual hybridized with EUB338 (25 ng, fluorescein labeled) and a mixture of all three PAO probes (Table 2; each 25 ng, CY3 labeled). Images were collected for fluorescein and CY3 channels, artificially colored, and superimposed. Arrowed yellow cells are the PAOs, since they are dual labeled with EUB338 (green) and PAO (red) probes. The bar for both panels C and D is 10 μm. (C) Mixed liquor from SBR A with operating data as given in Table 1. (D) Lightly sonicated mixed liquor from an EBPR SBR (ca. 10% P in the sludge) operating at 3.5% NaCl from a study of seafood-processing wastewater. Sludge kindly supplied by Nugul Intrasungkha. (E) Epifluorescence micrograph of GRC sludge (Table 1) dual hybridized with EUB338 (25 ng, fluorescein labeled) and PAO651 (Table 2; 25 ng, CY3 labeled). Separate images were collected for fluorescein and CY3 excitation, artificially colored, and superimposed. Standard-arrowed yellow cells are PAOs; diamond-ended-arrowed green-colored cells are other bacteria. The bar is for both panels and is 4 μm. (F) Methylene blue-stained image of the same field as in panel E. Standard-arrowed cells containing purple granules of polyphosphate are the same yellow cells in panel E. Diamond-ended-arrowed blue cells are the same green cells in panel E.

FIG. 3

Phylogenetic tree of near-complete 16S rDNA sequences obtained from sludges A, B, P, SBRH, SBR1, SBR2, and GC (Gold Coast, Queensland, Australia) determined in this study and sequences from publicly accessible databases. Rubrivivax gelatinosus (D16213) was used as the outgroup in analyses but is not shown in the tree. Evolutionary distance and parsimonious analyses were carried out in PAUP∗ employing 2,000 bootstrap resamplings. Closed circles at nodes indicate >75% bootstrap support from both analyses; open circles indicate 50 to 75% from both analyses; half-shaded circles are for analyses where one algorithm gave >75% bootstrap support and the other gave 50 to 75%. The coding indicates that the clone came from a hyper-P-removing sludge (ca. 15% P in the sludge) (P+++), a good P-removing sludge (P++), a fair P-removing sludge (P+), or a non-P-removing sludge (P−). The specificity of the published β-2 Proteobacteria probe (BTWO23a) and those of probes designed in this work (PAO probes and Rc988) are shown by solid lines. Dashed lines indicate that the probe does not have 100% identity with the indicated sequence. For example, Rc988 has one mismatch (at position 1009) with the SBRP112 sequence. In addition to specifically targeting the sequences indicated in the tree, probe BTWO23a also targets (with no mismatches) members of the β-3 and γ Proteobacteria, Iodobacter, Chromobacterium, Chromatium spp., and a green nonsulfur division clone, OPB9. The scale indicates 0.02 nucleotide change per nucleotide position. TCB, trichlorobenzene.

FIG. 4

Relationship between sludge P content (percentage of dry mass) and proportion of cells binding all three PAO probes as a percentage of the EUB338 probe-positive cells. Sludges evaluated included the Q (★), P (⧫), and S (●) sludges of Bond et al. (8, 10, 11), the GRC sludge at varying but stable P-removal efficiencies (▴), and the A sludge (■) (this study).