ABSTRACTThis study explores the metabolic implications of dual substrate uptake in“Candidatus Accumulibacter”, focusing on the co-consumption of volatile fatty acids and amino acids under conditions typical of enhanced biological phosphorus removal (EBPR) systems. Combining batch tests from highly enriched “Ca.Accumulibacter” cultures with conditional flux balance analysis (cFBA) predictions, we demonstrated that co-consumption of acetate and aspartate leads to synergistic metabolic interactions, lowering ATP loss compared to individual substrate consumption. The metabolic synergy arises from the complementary roles of acetate and aspartate uptake: acetate uptake provides acetyl-CoA to support aspartate metabolism, while aspartate conversion generates NADH, reducing the need for glycogen degradation during acetate uptake. We termed this type of metabolic interaction as reciprocal synergy. We further expanded our predictions to uncover three types of interactions between catabolic pathways when substrates are co-consumed by “Ca.Accumulibacter”: (i) neutral, (ii) one-way synergistic and (iii) reciprocal synergistic interactions. Our results highlight the importance of network topology in determining metabolic interactions and optimizing resource use. These findings provide new insights into the metabolism “Ca.Accumulibacter” and suggest strategies for improving EBPR performance in wastewater treatment plants, where the influent typically contains a mixture of organic carbon compounds.SynopsisThis research demonstrates how dual substrate uptake by “Ca.Accumulibacter” enhances metabolic efficiency in EBPR by reducing global ATP losses through optimization of storage polymer usage.