The effects of sequentially combined carbon (SCC) using a symbiotic relationship of methanol and acetic acid on biological nutrient removal were investigated in both the continuous bench scale process consisting of an anoxic, an aerobic and a final settling tank and intensive batch tests. Compared to the use of respective sole carbon sources, methanol and acetic acid, the use of SCC showed superior removal efficiency of nitrogen (98.3%) and phosphorus (approximately 100%). Furthermore, the use of SCC enhanced simultaneous denitrification and phosphorus uptake by denitrifying phosphorus removal bacteria (DPB), resulting in the highest specific denitrification rate (SDNR) of 0.252 g NO3-N/g VSS/d achieved from the first anoxic zone with methanol of 30 mg COD/l. From batch tests performed under carbon limited anoxic conditions, 1 g of nitrate was used by DPB for P-uptake of 1.19 g. According to this result, 0.205 g NO3-N/g VSS/d was accomplished by normal denitrifiers using methanol, and 0.047 g NO3-N/g VSS/d was achieved by DPB. This research also demonstrated that the increase of poly-β-hydroxybutyr ate (PHB) stored by phosphorus accumulating organisms (PAOs) could be of importance in improving aerobic denitrification. The use of SCC produced the highest P-release in the anoxic zone, indicating the amount of PHB would be higher compared to the use of other sole carbons. Therefore, the SCC could be a very effective carbon source for the enhancement of aerobic denitrification as well.

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