| Summary: | 博士 === 中興大學 === 環境工程學系所 === 95 === This dissertation aimed to investigate the biochemical characteristics of the isolated denitrifying phosphorus removal bacteria (DPB) which were able to remove nitrogen and phosphorus simultaneously. For screening procedures, determination of denitrification ability was suggested as the first priority. Isolated denitrifiers were then cultured under an aerobic-anoxic single-stage process by using both oxygen and nitrate as electron acceptors to examine P removal capacity. The denitrifiers with higher phosphorus removal efficiency in the log phase were chosen. On the contrary, the well-grown denitrifiers without obvious P removal were excluded owing to P release by considerable PHB formation. Most of the isolated DPBs had no anaerobic P release, but did have significant anoxic P uptake. Similar to the results observed from inoculation of enriched sludge, pure isolated DPB cultures showed no anaerobic P release but achieved N and P removal efficiencies of 96% and 86%, respectively. The isolated DPB strains were indentified as Brachymonas sp., Pseudomonas stutzeri, and Paracoccus denitrificans.
Paracoccus denitrificans PP15 was chosen as a model organism to investigate microorganism-mediated enhanced phosphorus chemical precipitation by varying Tris buffer concentrations. When Tris buffer was sufficiently provided up to 12 g/L, pH only increased from 7.0 to 7.4, and P. denitrificans PP15 removed only 10 mg P/L biologically. On the contrary, pH value increased to 8.5 without adding Tris buffer, and about 72 mg/L phosphorus was removed involved with biological and chemical mechanisms. When pH was higher than the breakpoint (pH 8.47) which was shown in the continuous pH curve via pH automatic online monitoring, phosphate concentration decreased suddenly from 108 to 55 mg P/L owing to chemical precipitation. Combined cation determination with powder X-ray diffractometer analyses, the major chemical precipitates in the environment were magnesium-phosphate compounds, used for P recovery. The strain could increase pH value spontaneously due to denitrification and acetate metabolism, and phosphorus removal was enhanced from 8 mg P/L (pH 7, pH controlled test) to 82 mg P/L (pH 8.8, pH-uncontrolled test), over 9-fold enhancement. The biological process treatment, accompanying with chemical precipitation, could remove COD, nitrogen, and phosphorus simultaneously, whereas chemical precipitation with pH increase might probably remove phosphorus only.
An anoxic condition, which was conducted with 2 g/L CH3COONa•3H2O (942 mg COD/L), 105 mg NH4+-N/L, and 15 mg P/L, had a better efficiency of P removal in Brachymonas sp. P12. Based on the results from a modified STS method for intracellular phosphorus extraction and fractionation, metal phosphates had the highest content with 10-13 mg P/g biomass. The sum of low molecular weight (LMW) polyP and high molecular weight (HMW) polyP increased to 10 mg P/g biomass. Compared to P uptake of 25 mg P/g biomass from bulk solution, about 33% of P uptake was for polyP accumulation. Repeated anoxic batch cultivations were conducted to examine the enhancement of polyP accumulating capacity after an initial anoxic preculture, and polyP concentrations were quantified by using a 31P NMR spectroscopy. Data from the repeated anoxic batch examinations indicated that polyP accumulation occurred in the log phase under anoxic conditions and the efficiency of polyP accumulation was not enhanced by repeated batch cultivations. In the second anoxic batch, polyP-accumulating enhancement was limited in an aging population even though sufficient nutrients were provided the same as the first batch. More new-generated cells and higher cell growth rates promoted higher proportion of polyP to P uptake. An increased polyP concentration in the log phase indicated that anoxic acetate metabolism improved polyP accumulation. Anoxic acetate metabolism of Brachymonas sp. P12 provided sufficient ATP for polyP accumulation, PHB formation, and cell growth, simultaneously.
The denitrifying phosphorus removal bacterium Brachymonas sp. P12 was used to investigate the enhanced biological phosphorus removal (EBPR) mechanism involved with polyhydroxybutyrate (PHB), glycogen, and phosphorus uptake in the presence of acetate under anoxic or aerobic conditions. The results showed that excess acetate concentration and aerobic cultivation could enhance PHB formation efficiency, and PHB formation might be stimulated by glycogenolysis of the cellular glycogen. The efficiency of the uptake of anoxic phosphorus was greater when PHB production was lower. The EBPR mechanism of Brachymonas sp. P12 for PHB, P and glycogen was similar to the conventional anaerobic–aerobic (or anaerobic–anoxic) EBPR models, but these models were developed under anoxic or aerobic conditions only, without an anaerobic stage. The anoxic or aerobic log phase of growth was divided into two main phases: (1) the early log phase, where acetate and glycogen were consumed to supply enough energy and reducing power for PHB formation and cell growth (P assimilation), and (2) the late log phase, which concluded the simultaneous degradation of PHB and remaining acetate for polyP accumulation. Glycogenolysis played a significant role in the alternate responses between PHB formation and P uptake under anoxic or aerobic conditions. For application of the denitrifying phosphorus removal bacterium Brachymonas sp. P12, aerobic cultivation would increase the level of PHB production, with anoxic cultivation further increasing P uptake.
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