Biosurfactant Solvent-Extraction Process and Characterization

The biosurfactants were recovered from the culture medium; solubilized extracellular biosurfactant is present in the supernatant and cell-bound biosurfactant is present in the suspended cell fraction. Initially, the culture medium was centrifuged at 8000 rpm for 10 min to separate cell pellets from the supernatant. The cell pellets were washed with 0.85% NaCl, while the supernatant was extracted with 10% (v/v) hexane to remove residual oil. The cell-bound biosurfactant was recovered by resuspending the cell pellets in methanol with shaking for 1 h. Although, most researchers use PBS to obtain cell-bound biosurfactants from cell pellets (Bustos et al., 2018), our preliminary results showed that almost all the biosurfactant in the cell pellet fraction could be extracted with methanol, while PBS (at pH 7.0 and 8.0) and chloroform gave lower yields (Supplementary Figure 3). The hydrophilicity of Weissella cibaria PN3 cell-bound biosurfactant was probably different from that of other LAB biosurfactants.

The cell-bound biosurfactant in methanol was extracted by an acid precipitation and solvent extraction method similar to the extraction method for extracellular biosurfactant in the supernatant. Briefly, the pH of the sample was adjusted to 2.0 with 6 M HCl to reduce the biosurfactant solubility before adding an equal volume of a chloroform and methanol mixture (2:1 v/v), and the solution was incubated in a rotary shaker at 200 rpm for 1 h (Khondee et al., 2015). The organic solvent was separated and evaporated by rotary evaporation. The viscous yellowish product was dissolved in methanol and filtered. The amount of crude biosurfactant was measured by weighing, while the crude biosurfactant yield was calculated as g/L based on the volume of the production medium. In this study, biosurfactant was not extracted from immobilized cells because residual oil and other bacterial metabolites accumulated on the carriers could be co-extracted with the biosurfactant, resulting in a product with high impurities.

Prior to characterization, the extracted biosurfactants were separated from impurities such as proteins and fatty acids following micelle-destabilization and ultrafiltration methods modified from Witek-Krowiak et al. (2011). Crude extracts of extracellular and cell-bound biosurfactants were dissolved in methanol to break up the micelles and diluted to 1.0 and 2.0 g/L, respectively. Concentrations lower than their critical micelle concentrations (CMCs) (Table 1) were selected to prevent the aggregation of biosurfactant molecules. Methanol solution containing biosurfactant monomers was filtered through a 5 kDa MWCO ultrafiltration membrane (Hydrosart Vivaflow200, Sartorius, United Kingdom). The biosurfactant monomers passed through the membrane into the permeate, while impurities were retained in the retentate. Methanol was removed from the permeate by evaporation at 40°C. After purification, biosurfactant weight decreased by 10–20%, while purified extracellular and cell-bound biosurfactant concentrations in PBS (pH 8.0) decreased to 0.8 and 1.9 g/L, respectively. The chemical composition of the purified biosurfactants were analyzed by the colorimetric method following Khondee et al. (2015). Total lipids, proteins and sugars were determined by sulfo-phospho-vanillin, Bradford assay and phenol-sulfuric acid, respectively. The functional groups of the purified biosurfactants were analyzed by Fourier transform infrared (FTIR) spectroscopy in ATR mode (Spectrum, GX, Perkin Elmer) at wavenumbers ranging from 4000 to 400 cm^–1 and a resolution of 0.3 cm^–1.

Comparison of the yield and characteristics of biosurfactants from Weissella cibaria PN3 with those from other LAB strains in a single production cycle.