Production of di-(2-ethylhexyl) phthalate by Bacillus subtilis AD35: Isolation, purification, characterization and biological activities

The emergence of extensive antibiotics resistant bacteria increased the demands for finding out new sources of antimicrobial agents. Marine niches were reported to be rich in many competent producers of significant bioactive compounds. On the course of screening program for new antimicrobials, a Bacillus strain was isolated from Alexandria sea shores, Egypt. According to the morphological, cultural and biochemical characteristics, 16S rRNA sequence analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), the strain was identified as Bacillus subtilis and designated as B. subtilis AD35. One phthalate derivative namely Di-(2-ethylhexyl) phthalate (DEHP) was purified from the crude extract of B. subtilis AD35 by high performance liquid chromatography (HPLC) and the structural elucidation of this compound was confirmed on the basis of gas chromatography-mass spectroscopy (GC-MS), Fourier infrared spectroscopy (FT-IR) and UV spectrum. The results of MIC of the purified DEHP were as follow: 16 μg/ml (Salmonella typhimurium), 32 μg/ml (Methicillin resistant Staphylococcus aureus, MRSA), 0.25 μg/ml (Listeria monocytogenes), 0.5 μg/ml (Aeromonas hydrophila), 8 μg/ml (Staphylococcus aureus), 4 μg/ml (Staphylococcus epidermidis), 4 μg/ml (Escherichia coli), and 8 μg/ml (Pseudomonas aeruginosa). DEHP produced by B. subtilis AD35 up to a concentration of 2500 μg/ml exhibited no cytotoxic effect against normal Vero cells. In addition, it did not show an antiviral activity against HAV or a significant growth inhibitory effect toward human colorectal adenocarcinoma and human mammary gland adenocarcinoma cell-lines.