Traditional Bordetella bronchiseptica vaccine production utilizes batch fermentation and complex medium. Cultivation parameters and medium composition have a direct impact on growth rate, biomass generation, and virulence factor production, specifically filamentoushemagglutinin (FHA). A high yield controlled large scale production process is desirable forvaccine production. In this study, statistical modeling was used to investigate the impact ofknown virulence modulators, MgSO4 and nicotinic acid. Lactate, succinate, citrate, and acetate were investigated as dual carbon sources in combination with glutamate to balancecarbon:nitrogen in medium. Steady-state and fed-batch fermentation were explored as potential cultivation techniques to increase yield. Statistical modeling yielded two optimalMg/SO4/nicotinic acid concentration conditions: 1 mM/0.1 mM/32.5 M and 0.1 mM/1.0mM/32.5 M. We also found the greatest yields from lactate-glutamate (commonly used inliterature) and acetate-glutamate. However, acetate-glutamate generated greater FHA and 7 times the biomass. Steady-state and fed-batch fermentation proved to be undesirablefermentation techniques.