Microbial flocculation is an aggregation phenomenon of bacterial cells in the form of flocs or flakes. In this study, we endowed ethanol producing E. coli KO11 cells with floc-forming ability by overexpressing a native bcsB gene (KO11/pNbcsB) without negative effect on ethanol production. Quantitative evaluation of the formed flocs was conducted on the basis of the protein amount included within floc structure. The results showed that glucose concentration and culture temperature were important parameters for E. coli floc formation. In particular, a temperature shift from 37 to 30 degrees C suppressed the floc formation at a negligible level and the shift to 40 degrees C doubled the amount of flocs. The sedimentation test confirmed that E. coli flocs completed sedimentation within 15 min after cessation of shaking while planktonic cells kept suspending. Finally, the repeated batch operation was performed to demonstrate the advantage of flocculating E. coli KO11. The amount of deposited cells in the culture of KO11/pNbcsB strain was about two times greater than that of original KO11 strain, thereby leading to a significant increase of ethanol productivity. In addition, the flocs lasted to be produced during the repeated batch operation, suggesting that the productivity will be enhanced in every operation.