On the spontaneous liquid-liquid phase separation during sophorolipids production: structural insights by small-angle X-ray scattering

Sophorolipids are well-known bio-based amphiphiles, commonly known as biosurfactants, extracted from the fermentation broth of the Starmerella bombicola.Sophorolipids are always a mixture of a closed lactone and open acidic forms, the former being in excess.Some of the keys to the success of sophorolipids since more than five decades is certainly their high titer after fermentation and the spontaneous transfer from the intra-to the extracellular environment.Sophorolipids spontaneously separate from the fermentation broth and sink to the bottom of the reactor, which simplifies the down streaming processing, contributing to increase the production at acceptable costs.However, this phenomenon is not systematic and directly impacted by fermentation parameters, although still poorly understood and generally explained by a demixing process, similarly observed in oil with water systems, and attributed to the more hydrophobic nature of lactonic sophorolipids.By employing a structural approach at the nanoscale (1 -500 nm) using advanced characterization tools like Small-Angle X-Ray Scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM) and cryo-scanning electron microscopy (cryo-SEM), this work provides a critical fundamental insight to the liquid-liquid sophorolipidwater phase separation phenomenon, which is demonstrated to be associated to a complex self-assembly process of a quaternary system composed of both acidic and lactonic sophorolipids (produced by S. bombicola), oil (substrate from fermentation) and water.The combination of SAXS, cryo-TEM and cryo-SEM unambiguously demonstrate that the bottom phase is characterized by a highly branched hydrated wormlike micelles network, while the top phase exclusively by water.The main parameter allowing to control this self-assembled 2 structure is the content of hydrophobic components, namely fatty acids and lactonic sophorolipids.