Physical enrichment of polyphosphate-accumulating organisms in activated sludge

Two methods that physically separate polyphosphate-accumulating organisms (PAO) from other organisms in activated sludge were developed. The first method used 4'6-diamidino-2-phenylindole dihydrochloride (DAPI) to selectively stain PAO. When excited with light at 340 nm, polyphosphate granules in DAPI-stained cells fluoresce yellow while cells without polyphosphate fluoresce blue. This difference in fluorescent response was used to separate PAO from non-PAO using flow cytometry. The second method consisted of a simple gradient centrifugation to physically separate PAO from non-PAO based on their density differences. Both methods produced cell suspensions with an increased PAO concentration. From an average PAO concentration of approximately 14% in a full-scale process, the DAPI-flow cytometry method produced sorted samples with PAO representing more than 70% of the total cells, while the density gradient method produced an approximate 43 to 48% PAO enrichment. The physical enrichment methods described herein should facilitate the identification and study of PAO that are relevant in full-scale enhanced biological phosphorus removal processes.