Statistical optimization for enhanced yields of probiotic Bacillus coagulans and its phage resistant mutants followed by kinetic modelling of the process

Probiotics are microorganisms which when administered in adequate amounts confer health benefits to the host. A leading pharmaceutical company producing Bacillus coagulans as a probiotic was facing the problem of recurring phage attacks. Two mutants viz. B. co PIII and B. co MIII that were isolated as phage resistant mutants after UV irradiation and MMS treatment of phage sensitive B. coagulans parental culture were characterized at functional and molecular level and were noted to have undergone interesting genetic changes. The non-specific genetic alterations induced by mutagenesis can also lead to alterations in cell performance. Hence, in the current study the parental strain and the two mutants were selected for shake flask optimization. Plackett-Burman design was used to select the significant culture variables affecting biomass production. Evolutionary operation method was applied for further optimization. The study showed wide variations in the nutritional requirements of phage resistant mutants, post exposure to mutagens. An increment of 150, 134 and 152 % was observed in the biomass productions of B. coagulans (parental type) and mutants B. co PIII and B. co MIII respectively, compared to the yield from one-factor-at-a-time technique. Using Logistic and modified Leudeking-Piret equations, biomass accumulation and substrate utilization efficiency of the bioprocess were determined. The experimental data was in agreement with the results predicted by statistical analysis and modelling. The developed model may be useful for controlling the growth and substrate consumption kinetics in large scale fermentation using B. coagulans.