End-product inhibition is a key factor limiting the production of organic acid during fermentation. Two rounds of heavy-ion beam irradiation may be an inexpensive, indispensable and reliable approach to increase the production of butyric acid during industrial fermentation processes. However, studies of the application of heavy ion radiation for butyric acid fermentation engineering are lacking. In this study, a second (12)C(6+) heavy-ion irradiation-response curve is used to describe the effect of exposure to a given dose of heavy ions on mutant strains of Clostridium tyrobutyricum. Versatile statistical elements are introduced to characterize the mechanism and factors contributing to improved butyric acid production and enhanced acid tolerance in adapted mutant strains harvested from the fermentations. We characterized the physiological properties of the strains over a large pH value gradient, which revealed that the mutant strains obtained after a second round of radiation exposure were most resistant to harsh external pH values and were better able to tolerate external pH values between 4.5 and 5.0. A customized second round of heavy-ion beam irradiation may be invaluable in process engineering.