Characterization Of Denitrifying Polyphosphate-accumulating Organisms (DPAOs) And Mechanism Of Simultaneous Nitrogen And Phosphorus Removal

Presently, nitrogen and phosphorus pollution which play an important role in controlling water pollution has received increasingly attention. In order to meet the current wastewater resources utilization, enhance the requirements of advanced treatment of wastewater, solve the inherent contradictions of traditional biological nitrogen and phosphorus removal process and deepen understanding the mechanism of denitrification and phosphorus removal, the startup had been systematic studied on the base of a sequencing batch reactor (SBR) type anaerobic/aerobic/anoxic process fed with domestic wastewater for simultaneous denitrification and phosphorus removal in present study. The screening, identification, the physiological and ecological characteristics and the nitrogen and phosphorus removal performance of DPAOs (denitrifying polyphosphate-accumulating organisms) was studied from the point of microbiology. Furthermore, bioaugmenation with DPAO was conducted in the rapid startup of the AOA SBR on that basis.The startup of SBR fed with domestic wastewater was investigated and the results indicated that the system had strong tolerance with lower C/P, despite the sustained reduction and fluctuation of the influent. The removal of COD, nitrogen and phosphorus achieved requirements basically and the startup was accomplished. The short-cut nitrification was realized during the startup, and the typical simultaneous denitrification and phosphorus removal process was occurred in anoxic phase. It mainly took NO2--N as an electron acceptor for phosphorus uptake. Moreover, the DPAOs utilizing NO2--N as an electron acceptor was enriched from 15.67% (DPAOs/PAOs) on day 1 to 41.5%(DPAOs/PAOs) on day 42 during the SBR startup.The DGGE analysis of 16S rRNA showed that the microbial community structure and population had an obvious succession during the startup. The main microorganism includedα-,β- andγ- Proteobacteria, Actinobacteria and Planctomeces. Meanwhile, Actinobacteria-related PAOs, Acinetobacteria and GAOs (glycogen-accumulating organisms) were enriched significantly. The DGGE analysis of nirS and nirK genes showed that, rest on nitrite reductase Nir, the denitrifiers was abundant, the community structure was stable however, which was not conformed with that of 16S rRNA.The phosphate uptake medium and the denitrification medium were adopt to isolate PAO (polyphosphate-accumulating organism) and denitrifying bacterium, and 14 strains were obtained totally. Screening by phosphorus uptake experiment, and assisted with nitrate reduction, metachromatic granules and PHB (poly-P-hydroxybutyrate) granules dyeing tests. The results indicated that 14 strains have the denitrification ability and the metachromatic granules and/or PHB granules, and can uptake phosphorus under aerobic condition. Identification results showed that strains of ZQP2, ZQP3, ZQN1, ZQN2, ZQN3, ZQN4, ZQN5, ZQN6 and ZQN7 belong to Bacillus sp., and strains of ZQP1, ZQP4, ZQP5, ZQP6 and ZQP7 belong to Pseudomonas sp.. The study on nitrite reductase Nir indicated that strains of ZQP2, ZQP3, ZQP4, ZQP7, ZQN2, ZQN3, ZQN4, ZQN5 and ZQN7 are nirS+ and nirK type (PCR results of the others were negative).The study of key growth factors showed that carbon source and pH were the most influences on growth and phosphorus uptake. The strains can get best growth under the condition of acetate or citrate as carbon source,6 to 9 of pH, and 25～35℃. It inhibited denitrifying phosphorus uptake when nitrate concentrations was more than 40mg/L. The effects of combination of two and three strains were not satisfactory, which did not have superiority as opposed to single-plant phosphorus uptake rate. Extracellular polymeric substances (EPS) of strain ZQN4 under aerobic condition were investigated. Experimental results suggested that protein, amounting to about 80% of total EPS content, was the major component in EPS of strain ZQN4, followed by DNA and carbohydrate. The extraction efficiency of EPS depends largely on the choice of extraction method. In this study, Ultrasonic method was effective and a good choice. The phosphorus removal efficiency of strain ZQN4 was approximately 89.4%, in which about 88% of the reduced phosphorus in the substrate was absorbed by strain ZQN4 while 12% of it was stored in the EPS, indicating the contribution of strain ZQN4 to phosphorus removal. Moreover, the most of reduced phosphorus existed in the form of phosphate PO43--P. Moreover, thekinetic models reflecting strain growth was constructed, which fitted well with growth and time and represented growth of the strain.By applying bioaugmentation with the addition of DPAO, the AOA SBR was started rapidly. The effluent of bioaugmented biological system was superior to that of the non-bioaugmented control, especially denitrification and phosphorus uptake. The effluent can reach the first class discharge standard A of urban sewage treatment factory (GB18918-2002) during the 13～16d of bioaugmented biological system.The DGGE analysis of 16S rRNA in bioaugmented system showed that the diversity of microbial communities was rich and bacterium crowd was in the process of dynamic change. The microorganisms mainly includedα- andγ- Proteobacteria, Flavobacteria, Pseudomonas and Bacillus, and Actinobacteria-related PAOs was dominant in population. The band represented the added strain ZQN4 always existed in the SBR during startup and formed a stable niche in the process of structural adjustment of microbial community.The DGGE analysis of nirS and nirK genes in bioaugmented system showed that, the structure of microbial community of nirS gene was more stable and population was more abundant when compared with that of nirK gene. Denitrifiers with nirS gene includedβ-Proteobacteria, Pseudomonas sp. and Rhodocyclus sp., while denitrifiers with nirK gene included Pseudomonas sp. and Rhizobium sp..The above studies illustrated that the bioaugmentation with DPAO addition can short startup time, improve performance of activated sludge to a certain extent and enhance wastewater treatment effect. It provides a guide and reference for application of bioaugmentation with the addition of DPAO, as well as an effective new way for efficient and stable treatment of biological nitrogen and phosphorus removal process.