3. Results and Discussion
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 oily characteristics. The microscopic results showed that biomass and broth were enclosed by the mixture of 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. 3). 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. 3). With the further decrease of O/S ratio (< 0.02 g/g), crystalline SLs gradually appeared in the broth (Fig. 3). 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 number 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 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 oil and SLs contents.
3.2. The mechanism 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. 4A), 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 oil content would significantly affect the settling time, thus exhibiting that O/S ratio was correlated to the average sedimentation rate (Fig. 4B). 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.
Stokes formula is used to calculate the sedimentation rate of spherical particles under an ideal condition. Although the SLs sedimentation is a complicated process and it is impossible to achieve an ideal environmental state, the Stokes formula can still give important theoretical guidance. According to formula 4, the sedimentation rate of spherical particles was mainly determined by the radius, density, and viscosity. Analysis of SLs particle size showed that when SLs was oily, the radius of SLs particle increased with the O/S ratio and the maximum radius maintained at 1.5 mm once the O/S ratio exceeded 0.04 g/g (Fig. 5A). In the real fermentation process, the O/S ratio was usually above 0.04 g/g, therefore the particle size of SLs seemed to be less influence on the SLs settling. In addition, the viscosity of fermentation broth was an important factor affecting the sedimentation rate of SLs. 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. 5B). 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 oil, the density decreased with the increase of O/S ratio (Fig. 5C). However, 1.0 g SLs was found to be bound to a maximum of 0.3 g oil (Fig. S2). Therefore, the minimum density of the mixture of SLs and 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 oil (Fig. 5D), and density difference was the main reason determining the SLs settling or floating. As the main reason for suspending SLs was the increase of oil concentration or bubbles, the density of the mixture of SLs and oil was lower than the supernatant to achieve temporary suspension, but SLs would be re-sedimented by standing still.
As mentioned above, the SLs sedimentation requires the formation of a mixture of SLs and oil. 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 increased of O/S ratio, the hydrophobicity of the mixture was strengthened, thus the settling rate was accelerated under gravity action.
In the settling process, the hydrophobic mixture formed by SLs and oil was the key to settling (Fig. 6). The stronger hydrophobicity, the faster sedimentation rate will be. In terms of sedimentation rate, it was mainly dependent on the density difference of supernatant and the mixture. 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 oil during the in-situ separation process. Especially for 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 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. S3A). Moreover, it could be found that after the treatment of ultrasound, the cell viability and SLs production capability would not be affected, demonstrating that it was feasible to introduce UEST to improve the efficiency of SLs sedimentation. (Fig. S3 B and C).
The average sedimentation rate of SLs enhanced with the increase of the ultrasonic time and power (Fig. 7A, B and C). The maximum sedimentation rate increase by 46.9% to 485.4% with UEST from high to low O/S ratios (Fig. 7 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 oil on viscosity, but also further reduces the loss of 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, and the sedimentation efficiency was related to supernatant density dependent on glucose concentration. 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 of in-situ separation of SLs were conducted with UEST once the SLs concentration in the broth reached 200-220 g/L, and the total of 2039.9 g SLs was produced with the consumption of 1545.7 g oil and 1979.3 g glucose respectively (Fig. 8A 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 and 0.58 g/g respectively (Fig. 8C). Moreover, UEST in-situ separation could reduce the losses of biomass, glucose and oil by 68.2%, 16.2%, and 65.5%, respectively (Table 1), in comparison to direct in-situ separation 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).
SLs has been regarded as a green and sustainable biosurfactants, but their high production costs limit large-scale application and development (Jiménez-Peñalver et al., 2019). Coupling fermentation on the basis of in-situ product separation is one of the most effective ways to reduce production costs and improve productivity (Table 2). Wang et al. (2020) designed a simple in-situ separation device coupled with the bioreactor and adopted high cell density fermentation to achieve high-efficient production of SLs. After running for 480 h, the productivity and yield of SLs were 2.39 g/L/h and 0.73 g/g, respectively, with an average separation efficiency of 74.3%. Zhang et al. (2018) developed a new bioreactor with dual ventilation pipes containing dual sieve plates (DVDSB) for semi-continuous fermentation of SLs. By regulating the air pressure and different sources of oil (transgenic or non-transgenic oil), the gravity sedimentation of SLs at the bottom of bioreactor could be accomplished with SLs titer, productivity, and yield of 477 g/L, 1.59 g/L/h and 0.60 g/g, respectively. Otherwise, Dolman et al. (2017) suggested that the glucose concentration could adjust the density of fermentation broth thereby controlling the ups and downs of SLs. The whole fermentation process lasted 1023 h to produce 623 g/L SLs, and during the SLs in-situseparation process, the recovery rate of SLs was as high as 86.0%, which was 9 times concentration of previously reported. Apart from SLs separation by gravity sedimentation, Liu et al. (2019) realized the SLs separation in the upper layer of the broth through the principle of froth flotation. Subsequently, part of biomass and glucose could be recovered by washing the settled SLs, and the final titer and productivity of SLs were 342 g/L and 1.55 g/L/h, respectively. Though these strategies have achieved relatively satisfactory results in the in-situ separation of SLs, the mechanism of SLs separation either by gravity sedimentation or by froth flotation was still not been clearly elucidated, which led to a blind separation process and low separation efficiency. Oil or glucose concentration has been pointed out to possess a significant impact on the settlement of SLs (Zhang et al., 2018), however, these researches focused more on the final results and failed to further analyze the causes of the sedimentation, resulting in big differences in different studies. Herein, the sedimentation mechanism of SLs was studied and it was found that the O/S ratio was the key factor affecting the SLs morphology and subsequent settling. In contrast, the sedimentation rate was dependent on the hydrophobicity of the oily SLs as well as the density difference with the broth. These results provided a solid foundation for rational regulation of SLs deposition. On the other hand, the UEST can accelerate the particle collision, make the particle become larger, eliminate the air bubbles, and further reduces the settling resistance (Luo et al., 2019). Palme et al. (2010) and Hincapié Gómez et al. (2015) applied ultrasonic enhanced deposition technology in the separation and recovery of microalgae and yeast cells, respectively. In this work, UEST was introduced to enhance the sedimentation of SLs, simultaneously greatly reduce the losses of oil, glucose, and biomass duringin-situ separation process. Moreover, semi-continuous fermentation by in-situ separation of SLs with UEST was conducted. Compared to the batch fermentation (168 h), the fermentation cycle was more than 2-fold longer (378 h), and the average productivity and yield from semi-continuous fermentation were increased by 26.5% and 23.4% respectively. This could be contributed to that the semi-continuous fermentation could separate out SLs and toxic substances in time, and allow the cells being in the optimal production environment, which also proved the feasibility of semi-continuous fermentation as a model for the efficient production of SLs. In comparison with other semi-continuous fermentations of SLs, both the productivity and yield in this work were at high levels, especially for the index of YSLs/DCW representing the SLs production yield to the DCW, its value reached 36.1 g/gDCW, which was 86.1% higher than the highest value in the literatures. Furthermore, the specific SLs productivity (QP/DCW) also exhibited a 41.2% higher than the highest value ever reported. It could be expected to achieve a more ideal production efficiency in combination with high cell density strategy, laying a foundation for further industrial application.