Polyhydroxyalkanoate (PHA)- and polyphosphate-accumulating traits of different taxonomic tetrad-forming organisms (TFOs) in two anaerobic-aerobic sequential batch reactors (SBRs) were characterized by the simultaneous use of fluorescence in-situ hybridization, PHB stain and DAPI stain. The two SBRs with glucose as the main carbon source were operated under different P:total organic carbon feeding ratios for more than 300 days, but both exhibited no enhanced biological phosphorus removal (EBPR) activity. Microscopic observations on sludge samples taken at various times from those two SBRs revealed that TFOs consistently accounted for more than 50% of total cells, and were mostly affiliated with the β- and γ-subclasses of Proteobacteria and the high G+C phylum of Gram-positive bacteria (HGC). Those TFOs from the β-Proteobcateria exhibited PHB stain positive and DAPI stain negative, indicating that they could utilize compounds other than polyphosphate (i.e. glycogen) as reducing power for PHA synthesis from glucose. In contrast, two types of TFOs within the HGC group showed negative PHB stain and positive DAPI stain, indicating their capacity to accumulate polyphosphate without the synthesis and degradation of PHA. This metabolic trait was different from the widely accepted biochemical model of EBPR and non-EBPR metabolisms. Other TFOs within the HGC group and γ-Proteobacteria showed negative responses to both PHA and DAPI stains, and their function in the deteriorated EBPR system need to be further clarified. Overall findings suggested that the phylogenic and physiological heterogeneity of TFOs in anaerobic-aerobic activated sludge systems were diverse and greatly exceeded the current understanding.

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