Two factor at a time approach by response surface methodology to aggrandize the Bacillus subtilis KLP2015 surfactin lipopeptide to use as antifungal agent

A Gram's positive, rod shaped and endospores-forming 'bacterial isolate KLP15′ was identified as Bacillus subtilis (Accession number KT459335) by the biochemical tests and 16S rRNA based sequencing. Lipopeptide(s) production from B. subtilis KLP2015 increased from 200.0 to 547.0 mg/l after the optimization of physico-chemical parameters like choice of nitrogen source, fermentation time, fermentation temperature and broth pH by 'One Factor at a Time' (OFAT) approach. Further, the yield of lipopeptide(s) increased to 985 mg/l from an earlier value of 547 mg/l when a "Two Factors at a Time (TFAT) approach using Response Surface Methodology (RSM) was attempted. The extracted crude lipopeptide fraction from the cell-free fermentation broth of B. subtilis KLP2015 was purified and characterized by High Performance Liquid Chromatography (HPLC), Thin Layer Chromatography (TLC), UV-Visible Spectrophotometry (UV-vis), Fourier Transform Infrared (FTIR) and MALDI-TOF studies. HPLC and TLC results confirmed the purity of the lipopeptide preparation and its Surfactin-type nature. The FTIR and MALDI-TOF spectra of the purified lipopeptide were found to be similar to that of an authentic Surfactin molecule. The purified lipopeptide showed potent 75.1% and 41.9% antifungal activity against Mucor sp. and Aspergillus niger, respectively on the basis of zone of inhibition of growth of these fungal strains. Moreover, the purified lipopeptide preparation of B. subtilis KLP2015 was most effective against the growth of Mucor sp. as reflected by the recorded MIC values of 6.25 µg/ml while MIC of lipopeptide for the A. niger was 12.5 µg/ml.