The return sludge sidestream process promotes phosphorus removal stability with synergistic activity of polyphosphate accumulating organism populations

Sidestream enhanced biological phosphorus removal (EBPR) is an alternative to conventional EBPR, although previous sidestream EBPR studies employ anaerobic sidestream tank (SST) hydraulic retention times (HRT) ranging from 1-60 hours. This study assesses how anaerobic fermentation time impacts polyphosphate accumulating organism (PAO) activity, where a suitable SST HRT found from fermentation tests was then applied in a sidestream EBPR process and its performance and microbial community were compared to conventional EBPR. The SST HRT maximising PAO activity was found to be 16 h, based on orthophosphate release rate and PHA storage. Bioreactors operated as either a conventional anaerobic/anoxic-aerobic (CONV) or return sludge sidestream (RSS) configuration for 180 days showed that the RSS process demonstrated greater long-term P removal stability, with a high and stable abundance of Tetrasphaera -related PAOs (15.9±6.9%) dominated by Phycicoccus elongatus . Accumulibacter was the main PAO in CONV EBPR (5.8±5.3%), although its unstable abundance likely explained CONV process instability, while the RSS showed stable Accumulibacter abundance (4.3±1.4%). Interestingly, the RSS system exhibited a higher abundance of glycogen accumulating organisms (GAOs) (30.4±8.6%), particularly from Micropruina (16.0±4.6%), while the CONV system showed low GAOs (3.5±1.9%), implying that GAOs did not negatively impact RSS performance. The contribution of Tetrasphaera and Accumulibacter to phosphorus removal in RSS was evaluated by Raman-FISH, showing that Accumulibacter resulted in 43% of the P removal, despite Tetrasphaera being >3.5 times more abundant. This study shows that the SST HRT applied in RSS led to stable P removal by a combination of two PAO groups, where the higher PAO diversity can mitigate GAO growth. • RSS can provide improved long term P removal stability over conventional EBPR • Sidestream tank HRT of 16 h was suitable for PAOs in RSS EBPR • Tetrasphaera contributed to 57% of P removal vs 43% by Accumulibacter via FISH-Raman • Abundant Competibacter and Defluviicoccus GAOs in RSS did not impact P removal • RSS stability due to synergistic P metabolisms of Tetrasphaera and Accumulibacter