The present work focuses on the investigation of denitrifying phosphorus removal organisms (DPB) in a novel two-sludge denitrifying phosphorus removal process by combining chemical with microbial analysis. When the two-sludge process operated stably over one year, good phosphorus (P) release and P uptake performance of activated sludge samples collected from this process were present in anaerobic and anoxic conditions, respectively, via batch test, showing that the ratio of P release specific rate to P uptake specific rate was 1.31. The analysis of energy dispersive spectrometry (EDS) showed that P content of activated sludge samples collected at the end of anoxic phase was 12.3% of dry weight, further demonstrating the existence of microorganisms responsible for phosphorus removal in this two-sludge process. From polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis, the presence of microorganisms mostly belonging to the phyla Firmicutes and Proteobacteria was observed, previously evidenced in the phosphorus removal wastewater treatment process. Fluorescence in situ hybridization (FISH) quantitative analysis showed that Accumulibacter responsible for phosphorus removal was dominant in this two-sludge process, accounting for 69.7% of all bacteria in activated sludge. These results obtained from chemical and microbial analysis in this study suggested that denitrifying phosphorus removal microorganisms were completely enriched in the two-sludge process proposed here. 1. Introduction Phosphorus (P) is one of the nutrients that can cause eutrophication of lakes, inland seas, and other natural waters, which threaten the safety of drinking water systems and ecological risk. Removal of P from wastewater is therefore important for preventing eutrophication. Nowadays, enhanced biological phosphorus removal (EBPR) has been broadly applied in wastewater containing phosphorus (P) treatment because of its lower cost and more environmental friendly technology compared to chemical precipitation or adsorption [1]. Different from chemical or physical treatment methods, EBPR, like other biological wastewater treatment systems, depends on the metabolism of microbial communities to remove P, possibly removing simultaneously organic or inorganic pollutants. Although EBPR system is an economic and environmental friendly technology for meeting the low P concentration in effluent, this process is difficult to control and is not always stable in the performance of P removal [2], probably due to a lack of understanding of microbiology in EBPR. So,
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