Aquatic environments are typically not homogenous, but characterized by changing substrate concentration gradients and nutrient patches. This heterogeneity in substrate availability creates a multitude of niches allowing bacteria with different substrate utilization strategies to hypothetically coexist even when competing for the same substrate. To study the impact of heterogeneous distribution of organic substrates on bacterioplankton, bioreactors with freshwater bacterial communities were fed artificial freshwater medium with acetate supplied either continuously or in pulses. After a month-long incubation, bacterial biomass and community-level substrate uptake rates were twice as high in the pulsed treatment compared to the continuously fed reactors even if the same total amount of acetate was supplied to both treatments. The composition of the bacterial communities emerging in the two treatments differed significantly with specific taxa overrepresented in the respective treatments. The higher estimated growth yield in cultures that received pulsed substrate inputs, imply that such conditions enable bacteria to use resources more efficiently for biomass production. This finding agrees with established concepts of basal maintenance energy requirements and high energetic costs to assimilate substrates at low concentration. Our results further imply that degradation of organic matter is influenced by temporal and spatial heterogeneity in substrate availability.