Optimizing resource use is essential for the survival and fitness of species in microbial communities ubiquitous in natural and engineered ecosystems. These ecosystems are often characterized by the simultaneous presence of multiple substrates such as volatile fatty acids, amino acids and sugars. Yet, the evaluation of metabolic potential for these microbial community members is predominantly based on single substrate utilisation. Metabolic and ecological implications of the interactions of multiple substrates, particularly in environments with changes in redox conditions and substrate availability, remain poorly understood. In this study, we investigate the metabolic interactions resulting from co-substrate utilization in polyphosphate-accumulating organisms within wastewater treatment systems. We combined experimental analysis of highly enriched “Ca. Accumulibacter” mixed cultures with genome-resolved metagenomics and conditional flux balance analysis (cFBA) to quantify the physiological relevance of co-substrate uptake. We observe that anaerobic co-substrate utilisation of acetate and aspartate result in metabolic interactions leading to optimized redox balance, reduced ATP losses and increased biomass yields by up to 8% compared to individual substrate use. Metabolic modelling revealed that these benefits emerge from the network topology, where the interaction of different metabolic routes gives rise to synergistic effects. Extending our analysis to additional substrate pairs, we classify metabolic interactions into three general types: (i) neutral, (ii) one-way synergistic and (iii) reciprocal synergistic. Our findings highlight the importance of metabolic interactions and cellular resource allocation strategies in dynamic microbial ecosystems. This study provides a broader ecological framework for understanding competitive metabolic strategies in environmental organisms. Co-substrate utilization can have direct implications for improving the yield or productivity of bioprocesses.