In microbial fuel cells (MFCs), exoelectrogens act as a catalyst and produce electricity directly. Exoelectrogens such as Geobacter in the heterotrophic condition acts as electron donors but when they transferred to the autotrophic condition, they act as electron acceptors and undergo continuous growth. However, most of the nonelectrochemical bacteria will collapse and disperse in the liquid phase with environmental change. In this study, we used electro-autotroph and acetate enrichment alternately rather than only an acetate-fed control system in the batch operation. After the five-batch operation, the microorganism diversity shifted from complex to simple, and Geobacter was the most abundant microorganism in the experimental system. The coulombic efficiency (CE) and the highest current density were 20%–45%, and 657 mA/m2, respectively, for the experimental group and 8%–20%, and 511 mA/m2, respectively, for the control group. The monosodium glutamate wastewater was treated in this study. The control group was acclimated in a shorter time, but the experimental group had higher current density and CE. The results indicate that the electro-autotroph process reduced the number of non-bioelectrochemical bacteria but induced the growth of exoelectrogens on the electrode, resulting in the MFCs achieving a higher efficiency.