3. Results
3.1. Effects of O/S ratios on SLs morphology and sedimentation
Under different O/S ratio conditions, SLs would exhibit different morphologies. When the O/S value was higher than 0.04 g/g, the SLs presented noncrystalline characteristics, definition of oily SLs. The microscopic results showed that biomass and broth were enclosed by the mixture of rapeseed oil and SLs due to their strong hydrophobicity. Under the action of gravity, SLs began to gradually precipitate, and the fermentation broth showed obvious stratification (Fig. 2). With the decrease of O/S ratio, the fermentation broth began to be in an emulsified state, and SLs no longer stratified with fermentation broth, thus presenting a relatively uniform state (Fig. 2). With the further decrease of O/S ratio (< 0.02 g/g), crystalline SLs gradually appeared in the broth (Fig. 2). By comparing the structure compositions of SLs in different forms, it could be found that under high O/S ratio condition (> 0.04 g/g), the proportion of deposited SLs, which was mainly a mixture of lactone- and acidic-forms SLs, accounted for 92% of the total SLs, while the supernatant only contained a small portion of lactone- and acidic- form SLs. In contrast, when the O/S ratio was low (< 0.02 g/g), the crystalline SLs composed of lactone-form SLs accounted for 38% of the total SLs, whereas the broth consisted of lactone- and acidic-form SLs (Table S1). These results were in agreed with the conclusion that acidic-form SLs has stronger hydrophilicity and lactone-form SLs has stronger hydrophobicity. Notably, the crystalline SLs could not settle on its own, but as the O/S ratio increased, it could be found through the microscope that the crystalline SLs would be surrounded by oily particles to form oily SLs, and then began to precipitate (Fig. S1). Therefore, it was further inferred that the settleability of SLs was dependent on their morphologies, which could be contributed to the hydrophobicity of lactone-form SLs as well as the rapeseed oil and SLs contents.
3.2. The characteristic of SLs sedimentation
When the O/S ratio was greater than 0.04 g/g, the sedimentation height was directly proportional to the concentration of SLs (Fig. 3A), and then the average sedimentation rate could be obtained by formula (1)-(3). According to formula (3), although the average sedimentation rate was related to SLs concentration in the broth, the rapeseed oil content would significantly affect the settling time, thus exhibiting that O/S ratio was correlated to the average sedimentation rate (Fig. 3B). With the increase of O/S ratio, the average of sedimentation rate of SLs was significantly enhanced, which reached the maximum value of 0.075 cm/s at 0.25 g/g. However, it started to decrease with the further increase of O/S ratio. As the main reason was the increase of rapeseed oil concentration, the density of the mixture of SLs and rapeseed oil was lower. Therefore, the average of sedimentation rate of SLs was decreased. Further increase of O/S ratio can achieve temporary suspension, but SLs would be re-sedimented by standing still.
It was found that the O/S ratio was the key factor affecting the morphology and subsequent settling of SLs. In general, the sedimentation rate of mixture (SLs and rapeseed oil) particles was mainly determined by the radius, density, and viscosity. Analysis of SLs particle size showed that when SLs was oily, with the increased of O/S ratio, the particle size of the mixture gradually increased, and the maximum radius was about 3.0 mm (Fig. 3C). Therefore, the particle size of mixture may be an important factor affecting on the SLs settling. In the early stage of fermentation, the viscosity of supernatant had marginal changes. When the SLs concentration was higher than 250 g/L, the supernatant viscosity was sharply increased (Fig. 3D). The high viscosity would affect the mixing and mass transfer, and reduce the production efficiency of SLs in the late fermentation. Therefore, when SLs concentration was around 200-220 g/L, the in-situ separation would be carried out in this study, thus viscosity had limited influence on SLs precipitation. By the density analysis, it was shown that after mixing SLs and rapeseed oil, the density decreased with the increase of O/S ratio (Fig. 3E). However, 1.0 g SLs was found to be bound to a maximum of 0.3 g rapeseed oil (Fig. S2). Therefore, the minimum density of the mixture of SLs and rapeseed oil was 1.10 g/cm3. In contrast, the density of the supernatant was mainly determined by glucose concentration, which was less than 100 g/L in real fermentation process, so the density of the supernatant was much lower than the mixture of SLs and rapeseed oil (Fig. 3F), and density difference was the main reason determining the SLs settling or floating. When the O/S ratio was low (< 0.04 g/g), crystallized or emulsified SLs would not settle by itself. When the O/S ratio was high (> 0.04 g/g), an oily mixture was formed and began to settle. With the further increase of O/S ratio, the hydrophobicity of the mixture was strengthened and the particle size increased, thus the settling rate was accelerated under gravity action.
In the settling process, the hydrophobic mixture formed by SLs and rapeseed oil was the key to settling (Fig. S3). The stronger hydrophobicity, the larger the particle size, the faster sedimentation rate will be. However, with the further increased of O/S ratio, density became the main influencing factor, so that the sedimentation rate of mixture decreased or suspension. In terms of sedimentation rate, it was mainly dependent on the particle size of mixture and density difference. To improve the efficiency of SLs separation and reduce the loss of substances during in-situ separation process, low glucose concentration and appropriate O/S ratio should be adopted.
3.3. Enhancement of sedimentation efficiency by UEST
Although SLs sedimentation could be achieved by adjusting the O/S ratio in the broth, it was common to result in some losses of biomass, glucose, and rapeseed oil during the in-situ separation process. Especially for rapeseed oil, its loss and SLs sedimentation efficiency always presented a contradiction, and it would be lost a lot at high O/S ratio of 0.25 g/g, even if SLs could be quickly settled. On the other hand, under the low O/S ratio condition, the rapeseed oil loss was reduced, but corresponding SLs sedimentation rate also slowed down. Therefore, UEST was introduced to accelerate the sedimentation rate of SLs and simultaneously reduce the loss of substrate and biomass. Ultrasound not only accelerates the aggregation of SLs particles, enhancing the gravity force, but also rapidly removes air bubbles, reducing the interference of air bubbles on SLs precipitation (Fig. S4 A). By comparing the effect of ultrasonic on particle size, it can be found that ultrasonic enlarged the particle size of mixture, increasing by more than 37.3% (Fig. S4 B). Moreover, it could be found that after the treatment of ultrasound, the cell viability and SLs production capability would not be affected (Fig. S4 C and D). Therefore, ultrasound can accelerate the deposition of SLs by reducing bubbles and increasing particle size, and has no significant effect on the growth of strain and SLs synthesis.
The average sedimentation rate of SLs enhanced with the increase of the ultrasonic time and power (Fig. 4A, B and C). The maximum sedimentation rate increases by 46.9% to 485.4% with UEST from high to low O/S ratios (Fig. 4 D). When the O/S was 0.10 g/g, the sedimentation rate reached 0.0165 cm/s, independent on ultrasonic power. Since low O/S ratio leaded to less loss in in-situ separation process, UEST could not only reduces the influence of rapeseed oil on viscosity, but also further reduces the loss of rapeseed oil and accelerates SLs separation under low O/S ratio condition. In the following experiments, the ultrasonic power of 100 W and the time of 10 min (the height of separation device was 10 cm and the sedimentation rate was 0.0165 cm/s) were adopted to achieve effective sedimentation.
3.4. Semi-continuous fermentation of SLs production by in-situ separation strategy with UEST
The SLs sedimentation was regulated by the S/O ratio. Therefore, before in-situ separation of SLs, the O/S ratio was adjusted to 0.10-0.12 g/g, and the glucose concentration was controlled at approximately 30 g/L. The whole semi-continuous fermentation cycle lasted 378 h, during which 4 times ofin-situ separation of SLs were conducted with UEST once the SLs concentration in the broth reached 200-220 g/L, and finally the total of 2039.9 g SLs was produced with the consumption of 1545.7 g rapeseed oil and 1979.3 g glucose respectively (Fig. 5A and B). Through the analysis of SLs productivity and yield during different phases, it was found that the cell activity has been maintained at a high level throughout the whole fermentation process and the average SLs productivity and yield reached 2.15 g/L/h and 0.58 g/g respectively (Fig. 5C). Moreover, UEST in-situ separation could reduce the losses of biomass, glucose and rapeseed oil by 68.2%, 16.2%, and 65.5%, respectively (Table 1), in comparison to direct in-situseparation strategy without UEST, thus achieving efficient SLs separation under low O/S ratio condition. Correspondingly, thein-situseparation efficiency and SLs separation rate improved by 34.5%% and 26.4%, respectively (Table 1).