Albie Gan, Gilda Carvalho, Liu Ye, Adrian Oehmen
Enhanced biological phosphorus removal (EBPR) processes have traditionally interpreted short-chain-length polyhydroxyalkanoates (SCL-PHAs) as the primary intracellular carbon storage polymers, largely due to the assumption that acetate and propionate are the dominant carbon sources. However, in fermentation-enhanced EBPR (F-EBPR) systems, such as return sludge sidestream (RSS)-EBPR, in-situ fermentation of return activated sludge can generate a broader substrate pool that may support more diverse intracellular carbon-storage pathways. This study demonstrates, for the first time, that long-chain-length PHAs (LCL-PHAs, >C14) dominate the intracellular PHA pool in RSS-EBPR sludge, accounting for 70.1 ± 5.0% of total PHAs, and exhibit the strongest association with phosphorus (P) removal performance (p < 0.05) among all intracellular storage compounds evaluated. Stoichiometric and pathway analyses suggest that β-oxidation is the preferred route for energy-efficient LCL-PHA synthesis, while the de novo pathway may serve as an alternative electron sink under highly reducing conditions. Genomic analyses identified Accumulibacter and Azonexus (formerly Dechloromonas) as the principal polyphosphate-accumulating organisms (PAOs) capable of LCL-PHA production through the β-oxidation pathway, whereas Tetrasphaera, Phosphoribacter, and Phycicoccus appear limited in this capacity due to the absence of essential biosynthetic genes. Overall, these findings challenge the conventional EBPR paradigm centred on SCL-PHAs and highlight the critical role of LCL-PHAs in carbon storage and P removal in sidestream EBPR, highlighting the need to incorporate LCL-PHAs into EBPR process monitoring, optimisation and future resource recovery strategies.