Various C₁–C₂ compounds are increasingly available through electrochemical reduction of CO₂. Although not always suitable as a sole substrate, these compounds can supplement a primary substrate like glucose to enhance microbial growth. Yet, the mechanisms underlying the effects of dual substrate consumption on growth rate and growth yield remain poorly understood. We developed a generalized, species-agnostic thermodynamic framework to partition anabolic and catabolic fluxes for various glucose/secondary substrate combinations, predicting maximum growth rate and growth yield as a function of the substrate ratio. The optimal strategy is to use the secondary substrate as electron donor, conserving the most efficient carbon source, glucose, for assimilation. Because many substrates yield similar energy per electron, biomass yield remains constant until glucose becomes limiting for anabolism. When further lowering the glucose fraction, additional assimilation of the auxiliary carbon source reduces the yield. The growth rate follows similar trends. Dual substrate growth enables generalists to produce more biomass from the total resource pool than a combination of specialists, conferring a competitive edge under substrate-limiting conditions. These theoretical observations align with experimental observations of lower residual substrate concentrations and dominance of generalists in natural and engineered oligotrophic environments. ... Read more