This work studied the dynamics of the biodiesel profile [as lipid-derived fatty acid methyl esters (FAMEs)] and microbial community diversity in activated sludge microbial consortia during the accumulation of lipid on short chain fatty acid (SCFA) as a carbon source. The model carbon source used was acetic acid buffered with sodium acetate; the nitrogen source was ammonium sulfate, and the feeding was done in fed-batch mode in bioreactors. The significant variability of FAMES in the grab-activated sludge was verified prior to bioreactor runs. Results showed that cultivation on acetic acid stabilized the profile of the FAMEs of activated sludge. This stabilization was likely due to proliferation of budding yeasts as confirmed by fungal diversity analysis. The dominant yeasts in the lipid-enhanced activated sludge were in the genera Williopsis. A confirmation that the quality of biodiesel improves through homogenization of the profile during the accumulation of lipid in activated sludge feeding on acetic acid reinforces the potential of SCFAs as alternative carbon sources in organic waste-to-bioenergy conversion.