Localization and identification of populations of phosphatase-active bacterial cells associated with activated sludge flocs

Abstract

The majority of phosphatase (PO4ase) activity detected in fresh aerobic activated sludge from a municipal wastewater treatment plant was associated with suspended floc material. PO4ase activity appeared to be localized in discrete bacteria-containing areas of the floc matrix based on the distribution of nucleic acid–stained cells and precipitated fluorescent crystals produced as a result of reaction of the enzyme(s) with the artificial substrate ELF™-PO4. Of the total floc-associated bacterial cells that stained positive with the nucleic acid–binding fluorochrome acridine orange (AO), 8.8 ± 1.2% displayed PO4ase activity based on the proximity of AO-stained cells to precipitated ELF crystals. Using a 16S rRNA oligonucleotide probe specific for the cytophaga–flavobacteria group, it was determined that 17–20% of the floc-associated bacteria that probed positive also displayed PO4ase activity. Furthermore, 35–45% of the ELF fluorescence was associated with bacterial cells that probed positive for the cytophaga–flavobacteria group. The results suggest that the cytophaga–flavobacteria, as a group, is important in mediating the liberation of inorganic orthophosphate (Pi) from phosphomonoesters of detrital organic phosphate (organic-P) in the aerobic activated sludge process of wastewater treatment.