Statistical Optimization of Fermentation Conditions for Sustainable cis,cis-Muconic Acid Biosynthesis by Pseudomonas putida

Many chemicals used in industry are produced using different isomers of muconic acid. The isomers are especially precursors for many industrially important polymers. In this study, eco-friendly production of cis, cis-muconic acid (ccMA) used in the production of polymers was carried out. Statistical experimental design was applied to optimize the production of ccMA by Pseudomonas putida KT2440 in batch culture. Plackett-Burman design was used to investigate the effect of 11 variables for ccMA production in the first optimization step. The results showed that ccMA production was most influenced by the initial pH of the medium and concentrations of Na-benzoate, NaCl, and (NH4)2SO4. After these significant factors were determined, the optimization of these factors was carried out with Response Surface Methodology (RSM) as the second optimization step. RSM showed that the optimal values of the factors (the initial pH and concentrations of Na-benzoate, NaCl, and (NH4)2SO4) were 7.96, 16.83 mM, 0.7 and 1.4 g/L, respectively. The other components of the fermentation medium were 18 g/L Na2HPO4, 10 g/L KH2PO4, 20 mM glucose, 1 mM MgSO4, 150 µM CaCl2, 13 µM FeSO4, and temperature of 35 oC. ccMA production increased about 4 times using statistical optimization methods.