2.4.3. Crude biosurfactant preparation
An aliquot of 20 mL of the cell-free fermented broth was col-lected and the pH was adjusted to 2.0 with 3 M HCl. This solutionwas maintained at rest for 12 h (overnight) at 4◦C for allowingthe precipitation of the biosurfactant; then it was centrifuged at10,000 × g and 4◦C for 15 min. This precipitate (crude biosurfac-tant) was dried (at 50◦C for 24 h), weighted [15,16] and thenstored for further analysis (surface tension, emulsification, stability,removal of contaminants and ecotoxicity). This recovery method isbased on the contact of H+ions and glutamic and aspartic acidsin the structure of surfactin, causing the protonation of the aminoacid side chain, then making it insoluble in an aqueous medium