This study highlighted the exploitation of mathematical models for optimizing the growth conditions that give the highest phosphatase productivity from a newfound Lysinibacillus sp. strain APSO isolated from a slime sample. Mathematical models facilitate data interpretation and provide a strategy to solve fermentation problems. Alkaline phosphatase (ALP) throughput was enhanced by 16.5-fold compared to basal medium based on a sequential optimization strategy that depended on two-level Plackett–Burman design and central composite design. The additional improvement for volumetric productivity and specific production yield was followed in a 7 L bench-top bioreactor to evaluate microbial growth kinetics under controlled and uncontrolled pH conditions. The pH-controlled batch cultivation condition neither supported cell growth nor enhanced ALP productivity. In contrast, the uncontrolled pH batch cultivation condition provided the highest ALP output (7119.4 U L⁻¹) and specific growth rate (µ = 0.188 h⁻¹) at 15 h from incubation time, which was augmented > 20.75-fold compared to the basal medium. To the authors’ knowledge, this study is the second report that deals with how to reduce the production cost of the ALP production process via utilization of agro-industrial waste, such as molasses and food waste (eggshell), as a nutrimental source for the improvement of the newfound Lysinibacillus sp. strain APSO ALP throughput.