"Candidatus Propionivibrio aalborgensis": A Novel Glycogen Accumulating Organism Abundant in Full-Scale Enhanced Biological Phosphorus Removal Plants

Abstract

Enhanced biological phosphorus removal (EBPR) is widely used to remove phosphorus from wastewater. The process relies on polyphosphate accumulating organisms (PAOs) that are able to take up phosphorus in excess of what is needed for growth, whereby phosphorus can be removed from the wastewater by wasting the biomass. However, glycogen accumulating organisms (GAOs) may reduce the EBPR efficiency as they compete for substrates with PAOs, but do not store excessive amounts of polyphosphate. PAOs and GAOs are thought to be phylogenetically unrelated, with the model PAO being the betaproteobacterial "Candidatus Accumulibacter phosphatis" (Accumulibacter) and the model GAO being the gammaproteobacterial "Candidatus Competibacter phosphatis". Here, we report the discovery of a GAO from the genus Propionivibrio, which is closely related to Accumulibacter. Propionivibrio sp. are targeted by the canonical fluorescence in situ hybridization probes used to target Accumulibacter (PAOmix), but do not store excessive amounts of polyphosphate in situ. A laboratory scale reactor, operated to enrich for PAOs, surprisingly contained co-dominant populations of Propionivibrio and Accumulibacter. Metagenomic sequencing of multiple time-points enabled recovery of near complete population genomes from both genera. Annotation of the Propionivibrio genome confirmed their potential for the GAO phenotype and a basic metabolic model is proposed for their metabolism in the EBPR environment. Using newly designed fluorescence in situ hybridization (FISH) probes, analyses of full-scale EBPR plants revealed that Propionivibrio is a common member of the community, constituting up to 3% of the biovolume. To avoid overestimation of Accumulibacter abundance in situ, we recommend the use of the FISH probe PAO651 instead of the commonly applied PAOmix probe set.

Keywords: Accumulibacter; EBPR; FISH; GAO; PAO; Propionivibrio; metagenomics.

FIGURE 1

Chemical transformations of a typical sequencing batch reactor (SBR)-cycle showing anaerobic volatile fatty acid (VFA) uptake, phosphorus (P)-release, glycogen degradation and polyhydroxyalkanoates (PHA) accumulation, followed by aerobic PHA degradation, P-uptake and glycogen replenishment.

FIGURE 2

Differential coverage plot of two metagenomes obtained from the SBR reactor at different sampling dates. Scaffolds are displayed as circles, scaled by length and colored by taxonomic classified essential single copy genes. Only scaffolds >5 kbp are shown.

FIGURE 3

Maximum-likelihood (PhyML) 16S rRNA gene phylogenetic tree of selected “Ca. Accumulibacter”-related sequences. Sub-group and clade classifications for “Ca. Accumulibacter”-affiliated sequences are taken from previous studies (He et al., 2007; Kim et al., 2010) and are given in parenthesis. Brackets (right) indicate coverage of probes designed in this study (blue text). Sequences representing genomes obtained in this study are given in orange text. The length of the alignment was >1200 bp. Bootstrap (from 100 analyses) branch-support values ≥50% are included: black circles, ≥90%; gray circles, ≥70%; white circles, ≥50%. Defluviicoccus vanus str. Ben 114^T (AF179678) served as the out-group. The scale bar represents substitutions per nucleotide base.

FIGURE 4

Maximum-likelihood phylogenetic tree of polyphosphate kinase gene (ppk1) genes from selected Accumulibacter-related organisms. Sub-group and clade classifications for Accumulibacter-affiliated sequences are taken from previous studies (He et al., 2007; Peterson et al., 2008; Mao et al., 2015) and are given in parenthesis. Sequences representing genomes obtained in this study are given in orange text. Sequences were aligned in MEGA6 applying a Muscle alignment with default settings. Sequences were trimmed giving an alignment length of 1006 bp. Bootstrap (from 100 analyses) branch-support values ≥50% are included: black circles, ≥90%; gray circles, ≥70%; white circles, ≥50%.

FIGURE 5

Phylogenetic position of the genomes from the family Rhodocyclaceae in the reference genome tree generated by CheckM. The CheckM tree is inferred from the concatenation of 43 conserved marker genes and incorporates 2052 finished and 3604 draft genomes from the IMG database (Parks et al., 2015). Sub-group and clade classifications for “Ca. Accumulibacter”-affiliated sequences are taken from the articles describing the genomes and are given in parenthesis. Sequences representing genomes obtained in this study are given in orange text.

FIGURE 6

Composite FISH micrographs of activated sludge from the (A) lab-scale reactor and (B) the full-scale WWTP at Tarm, Denmark. In both images “Ca. Propionivibrio aalborgensis” cells appear white having hybridized with Prop207 (green), PAOmix (red) (A) PAO462 + PAO846; (B) PAO462 + PAO846 + PAO651) and EUBmix (blue); “Ca. Accumulibacter sp.” appear magenta with their cells hybridizing the PAOmix and EUBmix probe sets; all other cells hybridized with EUB mix only and appear blue. Scale bar represents 20 μm.

FIGURE 7

Fluorescence in situ hybridization (FISH) micrographs of the Nile blue A stained reactor biomass at the end of the anaerobic period. All images are of the same field of view. (A) FISH image with the PAO651 probe (Cy5 – blue) targetting “Ca. Accumulibacter”; (B) FISH image with the Prop207 probe (FLUOS – green) targetting “Ca. Propionivibrio aalborgensis”; (C) Nile blue A stain. PHA granules appear red; (D) composite image of (A-C). Yellow cells, “Ca. Propionivibrio aalborgensis” with PHA inclusions; Magenta cells, “Ca. Accumulibacter” with PHA inclusions. Scale bar represents 20 μm.

FIGURE 8

Fluorescence in situ hybridization micrographs of the 4′,6-diamidino-2-phenylindole (DAPI) stained reactor biomass at the end of the aerobic period. All images are of the same field of view. (A) FISH image with the PAO651 probe (Cy5 – blue) targetting “Ca. Accumulibacter”; (B) FISH image with the Prop207 probe (Cy3 – red) targetting “Ca. Propionivibrio aalborgensis”; (C) DAPI stain. PolyP granules appear yellow; (D) composite image of (A-C). Scale bar represents 20 μm.

FIGURE 9

Diagrammatic representation of selected carbon and energy transformation pathways encoded by the “Ca. P. aalborgensis” genome. POR, pyruvate synthase (PROAA_v1_2850001-3); PDC, pyruvate dehydrogenase complex (PROAA_v1_1570009-11); CS, citrate synthase (PROAA_v1_2080008); ACN, Aconitase (PROAA_v1_510025; 170008); ICL, Isocitrate lyase (PROAA_v1_1090035); MS, Malate synthase (PROAA_v1_110003-4: fragmented); ICD, Isocitrate dehydrogenase (PROAA_v1_1090020); OGD, 2-oxoglutarate decarboxylase (PROAA_v1_2080005-7); SCS, Succinyl-CoA synthase (PROAA_v1_280019-20); SDH, Succinate dehydrogenase complex (PROAA_v1_1090002-5); FRD, Fumarate reductase (PROAA_v1_1190017-20); FH, Fumarate hydratase (PROAA_v1_250024); MD Malate dehydroganse (PROAA_v1_1090008); MCM, Methylmalonyl-CoA mutase (PROAA_v1_1920004); PCC, Propionyl-CoA carboxylase (PROAA_v1_70002; 1920002); MCD, Methylmalonyl-CoA decarboxylase (PROAA_v1_1060015-18); RBC, Ribulose biphosphate carboxylase (PROAA_v1_1050003); PhaC, Polyhydroxyalkanoate synthase (PROAA_v1_130029; 530016; 970001); PhaZ, Polyhydroxyalkanoate depolymerase (PROAA_v1_2430001); ACP, Acetate permease symporter (PROAA_v1_2930003); AckA, Acetate kinase (PROAA_v1_630017); Pta, Phosphate acetyltransferase (PROAA_v1_630016); Acs, Acetyl-CoA ligase (PROAA_v1_250023); PrpE, Propionyl-CoA ligase (PROAA_v1_2840001); F₁F₀ ATP synthase (PROAA_v1_510003-10); HOX, Type 3d bidirectional NAD(P)-linked hydrogenase (PROAA_v1_1030015-8); PPC, Phosphoenolpyruvate carboxylase (PROAA_v1_1450023); PEP, Phosphoenolpyruvate. EMP, Embden-Meyerhof-Parnas.