The study investigated the effects of the feeding regime on the substrate metabolism under aerobic conditions. Throughout the study, two parallel sequencing batch reactors were operated using either short-term (pulse) or long-term (almost continuous) feeding of acetate at two different sludge ages of two days and eight days. The microbial characterization studies showed that the feeding regime did not change the microbial composition as determined by the fluorescent in situ hybridization (FISH) analysis, however did strongly affect the substrate utilization mechanism. Additionally, the same microbial culture was able to utilize acetate with or without poly-beta-hydroxybutyrate (PHB) storage under pulse or continuous feeding, respectively. Conversely, the selected sludge ages induced significant changes in the microbial composition and floc structure, however, the well settling and bulking biomass selected at the sludge ages of eight days and two days, respectively, did not significantly affect the substrate utilization mechanism, where storage or growth prevailed as a result of either pulse or continuous feeding.