2.2. SLs sedimentation mechanism
2.2.1. Influences of rapeseed oil to SLs ratios on SLs morphology and sedimentation
The ratio of rapeseed oil to SLs (O/S) (0.075, 0.10, 0.13, 0.15, 0.18, 0.20, 0.22, 0.23, 0.25, 0.30 g/g) was quickly detected and regulated by the low-field nuclear magnetic method.[19]SLs morphology was observed by optical microscopy (BMDH200 microscope, Sunny Optical Technology Co., Ltd), and the main structures of SLs with different morphologies were analyzed by LC-MS descripted as Chen.[7]
Furthermore, 10 mL of the fermentation broth with different O/S ratios were processed by standing still to observe the SLs sedimentation, and the volume of lower layer was recorded at different time points until it was unchanged during 2 h.
2.2.2. Measurements of broth viscosity and SLs particle size
Twenty milliliters of broth with different O/S ratios were obtained to measure the viscosity of supernatant with a viscosity meter (Brookfield DV-Ⅱ). Another 5 mL broth was thoroughly mixed, and the SLs particle size was analyzed by nanometer size and ZETA potential analyzer (Beckman Coulter Nano-ZS).
2.3. Effect of ultrasound assistance on SLs precipitation
2.3.1. Effect of ultrasonic ultrasound assistance on cell activity, SLs production, bubble and particle size
An appropriate amount of fermentation broth was obtained and treated at different ultrasonic times (0, 10, 20, 30, 40, 50, and 60 min) and power (100, 200, and 300 W) in a 10 L ultrasonic cleaning machine (SHT-70al, 40 kHz). And then, the treated broth was diluted at appropriate times and coated on the plate to observe the colony-forming unit (CFU) after 72 h. Otherwise, after different ultrasonic treatments, the cells from 50 mL fermentation broth were concentrated by centrifugation, resuspended, and inoculated into a medium only containing glucose and rapeseed oil in a 500 mL shake flask with 50 mL working volume to investigate the SLs production capacity.
An appropriate amount of fermentation broth was obtained and treated at 100 W power in a 10 L ultrasonic cleaning machine, and record the level of fermentation broth. In addition, An appropriate amount of fermentation broth(O/S=0.1) was obtained and treated at different ultrasonic power (0, 100, 200, and 300 W) for 5 s, and detected particle size.
2.4. Semi-continuous fermentation based on ultrasonic assisted treatment
In semi-continuous fermentation, the optimal O/S ratio was adopted to rational control the sedimentation of SLs for in-situ separation. According to our previously developed SLs separation equipment, the improved in-situ separation platform of UEST was shown in Fig 1.[20] The fermentation broth was rapidly pumped into the separation unit, which was placed in the ultrasonic machine, by a peristaltic pump at 5 mL/s. The ultrasonic power and time were controlled at 100 W and 10 min. Subsequently, the SLs was intermittently discharged from port c for 1 min, and the supernatant was refilled from port b to the fermenter until the SLs concentration in the fermenter was lower than 60 g/L. Finally, the separated crude SLs was washed by 1/2 proportion of sterile water (v/v), and thoroughly mixed by stirring. After standing for 20 min, the supernatant was pumped back to the fermenter to recover the biomass, glucose and rapeseed oil. Due to the loss of biomass after the operation of SLs in-situ separation, medium and sterile water were replenished to the initial volume of 2.5 L.