Higher order volatile fatty acid metabolism and atypical polyhydroxyalkanoate production in fermentation-enhanced biological phosphorus removal

Enhanced biological phosphorus removal (EBPR) is an established wastewater treatment process, but its wider implementation has been limited by factors like high temperature and low carbon availability. Fermentation-enhanced EBPR (F-EBPR) processes have shown promise in addressing these limitations, but the underlying mechanisms are not fully understood. This study investigates the metabolism of higher order (C_4–5) volatile fatty acids (VFAs) in F-EBPR systems using a combination of carbon isotope labelling and shotgun metagenomic sequencing analyses. Results show that butyrate (HBu) uptake leads to the formation of both typical (C_4–5) and atypical (C₆₊) polyhydroxyalkanoates (PHAs) through a combination ofβ-oxidation and standard condensation pathways, while the putative role of HBu oxidisers were identified relative to substrate composition in F-EBPR processes. Metagenomic analysis reveals the presence of genes required for higher order VFA metabolism in both polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs). This study also highlights the limitations of current models in describing F-EBPR processes and emphasises the need for improved models that account for higher order VFA metabolism and microbial community dynamics.