Production optimization of antifungal lipopeptides by Bacillus subtilis CtpxS2-1 using low-cost optimized medium

Abstract

The aim of the present study was evaluated the use of low-cost optimized medium defatted soy flour and molasses (DSF40–MOL5; 40 g l−1 defatted soy flour, 5 g l−1 molasses plus mineral trace supplements) to improvement the antifungal lipopeptides production by biocontrol agent Bacillus subtilis CtpxS2-1. First, population dynamics of CtpxS2-1 in DSF40–MOL5 medium was recorded in a 5-l fermenter during 24 h, 48 h and 72 h and compared with the bacterium grown in medium optimal for lipopeptide production (MOLP) as control. Results showed that the CtpxS2-1 population dynamics, pH and oxygen consumption was similar in both media with a 4–h lag phase, followed by exponential growth and maximum production around 9.5 log CFU ml−1 after 16–18 h. Second, lipopeptides extracts quantifications, followed of high-performance thin layer chromatographic (HPTLC) and bioautography analysis against Colletotrichum acutatum showed a significant increment of fengycin and iturin lipopeptides production and their antifungal activity in DSF40–MOL5 compared with MOLP. PCR (RT-PCR)/RT q-PCR analysis during production confirmed the transcription of fengycin, iturin an surfactin lipopeptides by amplification of cDNA; and that FenB and ItuA genes were progressive expressed in higher amounts throughout in DSF40–MOL5 than MOLP. The biosynthesis of FenB gene and its antifungal activity against C. acutatum was the highest for CtpxS2-1 grown in DSF40–MOL5. Surfactin genes were also expressed during production process however their didn’t showed antifungal activity Finally, treatment of artificially infected seed of three cultivars of Lupinus mutabilis with CtpxS2-1 grown in DSF40-MOL5 reduced anthracnose up to 0% and improved seed and plant health. This evidence supports that antifungal potential of B.subtilis CtpxS2-1 to control lupin anthracnose could be enhanced using low-cost medium DSF40–MOL5 and it could provide a reliable method for scaling up the bacterial fermentation process at industrial level.