Shelf-life enhancement of bio-inoculant formulation by optimizing the trace metals ions in the culture medium for production of DAPG using fluorescent pseudomonad R62

Statistical experimental design was used to optimize the concentration of trace elements for production of antifungal compound, 2,4-diacetylphloroglucinol (DAPG), from fluorescent pseudomonad R62 in shake-flask cultivation. The selection of the trace metal ions, influencing DAPG production, was done using Plackett-Burman design (PBD). Only Zn(2+), Mn(2+) and MoO(4)(2-) were the most significant components (p<0.05). A quadratic model was used to fit the response. Application of response surface methodology (RSM) revealed that the optimum values of the salts of the trace elements Zn(2+) (ZnSO(4)·7H(2)O), Mn(2+) (MnCl(2)·4H(2)O), and MoO(4)(2-) (Na(2)MoO(4)·2H(2)O) were 83, 42 and 135μM, respectively, to achieve 125 mg/L of DAPG, which was nearly 13-fold more compared to its production in basal synthetic medium in shake flask. The studies in 14L bioreactor resulted in 135 mg/L of DAPG at the end of 36 h of cultivation. The culture broth containing 125 mg/L of DAPG was found to be sufficient for keeping the bio-inoculant viable in non-sterile talcum powder-based formulations (which contained 25μg DAPG/g carrier) when stored at 28°C for 6 months. The structure of the purified DAPG was confirmed using (1)H NMR and mass spectrometry.