1. Introduction
Sophorolipids (SLs), which are regarded as one of the most promising biosurfactants, have been widely used in cosmetic, food, pharmaceutical fields due to their low toxicity and high biodegradability (Jahan et al., 2020; Van Bogaert et al., 2007). Many microorganisms can synthesize SLs, includingCandida(Gaur et al., 2019), Pichia(Thaniyavarn et al., 2008), andRhodotorula (Sen et al., 2017). Candida bombicola is the most widely adopted specie due to its high efficiency of SLs production (Zhou et al., 2019). SLs are the mixtures of many structural derivatives consisting of two parts, a hydrophilic sophorose and a 16-18 carbon hydrophobic hydroxyl fatty acid with general 16-18 carbon atoms (Fig. 1) (Chen et al., 2020). The differences of SLs structures are dependent on the degree of acetylation, the length of carbon chain, the unsaturation of fatty acids, and internal esterification, which further determined the properties of SLs (David et al., 2003; Hu and Ju, 2001; Jimnez‐Pealver et al., 2020). Therefore, SLs fermentation process is a complex multiphase system of gas, liquid, and solid, not only containing SLs with different structural properties (solid crystallization, oily, etc.), but also possessing bubbles, lipids, glucose, cells, and other substances in the fermentation broth. This poses a great challenge to the effective regulation and optimization of the fermentation process.
Biosurfactants have the advantages of green production and environmental friendliness, compared with chemical surfactants. However, the high production cost of SLs is still the main factor that hinders their development and application (Ashby et al., 2013; Ma et al., 2020). Therefore, it is necessary to develop adequate process control strategies to improve production efficiency. To date, great progress has been made on SLs optimization in batch fermentations. However, the raise of SLs concentration during fermentation leads to an increase in viscosity, which further results in the difficulty in mixing and mass transfer, and finally causing a sharp decline in fermentation efficiency (Jia et al., 2017). Semi-continuous fermentation basing on in-situ separation of products can segregate part of SLs from the fermentation broth, thereby improving the rheological characteristics and maintaining efficient synthesis of SLs. Different types of devices have been developed to improve the efficiency of in-situ separation (Wang et al., 2019). In this regard, gravity sedimentation is the main method for in-situ separation of SLs. Zhang et al. (2018) found that the density of the fermentation broth could be adjusted by controlling the glucose concentration, so as to achieve the sedimentation of SLs. Subsequently, genetically modified soybean oil was used to achieve the sedimentation and separation of SLs. Although these studies have achieved good results, the mechanism of SLs sedimentation in a heterogeneous fermentation system has not yet been clearly elucidated, thus it is difficult to rationally control SLs sedimentation and separation.
In general, a large number of cells is lost during SLs in-situ separation process, so the efficiency of subsequent SLs synthesis will be significantly affected. Moreover, SLs have a strong affinity with oil, which also leads to the loss of oil, thereby reducing the substrate conversion rate. On the other hand, the improvement of separation efficiency also contributes to the efficient production of SLs. Ultrasonic enhanced sedimentation technology (UEST) consists of the use of particle agglomeration by acoustophoresis, which causes increased gravitational force from larger effective particle size to increase the sedimentation rate. UEST has been applied in improving microalgae production, sludge settling and dehydration efficiency (Hincapié Gómez et al., 2015; Maddikeri et al. 2015). Otherwise, Palme et al. (2010) reported that UEST was introduced in SLs fermentation for in-situ separation of cells, and the cell recovery was up to 99.0% without affecting the cell activity. However, it should be noted that only 10% of SLs could be recovered by this strategy.
In this study, the influences of oil concentrations on SLs morphology and sedimentation were investigated, and then the mechanism of SLs sedimentation was further analyzed. On this basis, UEST was introduced for in-situ separation of SLs, and finally a rational semi-continuous fermentation process was developed for highly efficient SLs production.