Response surface methodology mediated optimization of medium components for growth density and rate of mycoplasma gallisepticum

This study aimed to optimize the composition of a liquid medium for Mycoplasma gallisepticum growth rate and density through statistical approach. The growth concentration of M. gallisepticum was determined by plotting a standard curve using Real-Time Quantitative PCR. The one-factor-at-a-time method and the Plakett-Burman design were initially applied to identify the factors that influenced the biomass of M. gallisepticum. The steepest ascent experiment and response surface methodology (RSM) with Box-Behnken design was employed to simultaneously assess the effects of multiple factors. Finally, optimization of the initial pH and validation of the model were done. The optimum concentrations of the critical components were obtained as follows: 203.96 mL/L of horse serum, 9.64 g/L of glucose, and 9.49 g/L of PPLO broth. The nucleic acid copy number of M. gallisepticum reached 1010.5147 copies/mL and the viable cell count reached a maximum of 109.8451 CCU/mL. This medium reduced the incubation time by approximately 6 h, and M. gallisepticum nucleic acid concentration and viable cell count were higher than those in the modified Frey medium (9.99 and 7 times, respectively). The new liquid medium is likely to improve productivity and reduce the production costs for vaccine-manufacturing companies in the future by reducing incubation times and increasing the growth rate and concentration of M. gallisepticum.