Fuel ethanol is highlighted for being renewable and carbon neutral, but a multi-scale challenge exists that hampers its role as a future energy substitute, and with the breakthrough of cellulose pretreatment and enzyme hydrolysis technology, fermentation itself has become one of the main limiting factors for process enhancement. To tackle the problem, this paper studies periodic operation strategies to improve process performance and avoid possible instabilities. Firstly, superiority by periodic operation over the corresponding steady-state operation is verified. Then, to settle the problem of possible instabilities under impulsive control, stability of state-impulsive control is conducted and for the studied case, any self-cycling fermentation starts from batch mode with 50 % discharge-and-refill portion exhibits superior repeatability property, which allows continuous fermentation to operate under very slow overall dilution rate to cope with the extreme slow fermentation rate. Finally, on-off operation problem is formulated that is governed by a multidimensional nonlinear system, affine to the manipulated variable that is under the isoperimetric constraint; with the use of Pontryagin maximum principle, the optimal synthesis of the optimal strategies under integral constraint is computed out, and a practically plausible on-off operation strategy for ethanol fermentation is formulated with guaranteed periodic stability and optimality.