2. Material and methods
2.1. Microorganism, media, and culture conditions
C. bombicola ATCC 22214 was obtained from the Guangdong Culture
Collection Center (China) and stored at -80℃ in 20% glycerol solution.
The seed medium consisted of 50 g/L glucose, 1 g/L
KH2PO4, 4 g/L
(NH4)2SO4, 0.5 g/L
MgSO4·7H2O, and 10 g/L corn steep liquor
(CSL). The seed was cultured in a 1 L baffled shake flask
with
200 mL working volume at 200 rpm and 25℃ for 48 h.
The initial fermentation medium in a 5 L bioreactor (Shanghai Guoqiang
Bioengineering Equipment Co., Ltd., China) consisted of glucose 100
g/L,
KH2PO4 1 g/L,
(NH4)2SO4 4 g/L,
MgSO4·7H2O 0.5 g/L, CSL 10 g/L. All
culture media were sterilized at 115℃ for 30 min. The initial volume of
2.5 L with an inoculum of 3% was cultured at 25℃ for 168 h. Aeration at
0.5 vvm and dissolved oxygen (DO) above 30% of the saturation
concentration were maintained by adjusting the agitation in a stepwise
manner. A pH of 3.5 was maintained by addition of 4 M NaOH solution,
during the whole process. The fed-batch fermentation cycle was 168 h.
During fermentation, glucose concentration in the broth was maintained
at 30-80 g/L. Oil supplementation
rate was 0.5 g/L/h for the first 24 h, after which oil concentration was
maintained at 2-15 g/L, according to oil consumption. The
semi-continuous fermentation period was 378 h, and SLs was
intermittently separated by the separation unit every 72 h. The
supplementation rate of glucose and rapeseed oil was optimized according
to the sedimentation rate of SLs. After every two in-situseparations, 1/5 of the initial nutrition was supplemented.
2.2. SLs sedimentation mechanism
2.2.1. Influences of oil to SLs ratios on SLs morphology and
sedimentation
The ratio of 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
(Chen et al., 2019).
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 (Chen et al., 2020).
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 hours.
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 and
SLs production
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 cell activity. Otherwise, the
strains after different ultrasonic treatments were concentrated by
centrifugation, resuspended, and inoculated into a medium contained only
glucose and oil (50 mL of fermentation broth strains were inoculated
into a 500 mL shake flask with 50 mL working volume). And compared
strain growth and SLs production.
2.4. Semi-continuous fermentation
based on ultrasonic assisted treatment
In semi-continuous fermentation, the optimal O/S was adopted to rational
control the sedimentation of SLs for in-situ separation.
According to our previously developed SLs separation equipment (Liu et
al., 2019), the improved in situ separation platform was shown in Fig 2.
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 oil. Due to the loss of biomass after the operation of SLsin-situ separation, medium and sterile water were replenished to
the initial volume of 2.5 L.
2.5. Analytical methods
Glucose concentration was analyzed by an enzymatic bio-analyzer
(SBA-40C, Shandong Academy of Sciences, China). Oil concentration in the
broth was quickly determined by the low-field nuclear magnetic method
(Chen et al., 2019). For oil content determination, three parallel broth
samples were extracted twice, using the same volume of n-hexane. The
upper layer was transferred to another tube and dried in an oven for 24
h, until constant weight. The bottom layer was washed twice with alcohol
and dried at 80 ℃ for 24 h to measure dry cell weight (DCW). The
concentrations and structures of SLs were determined by HPLC and
HPLC-MS, respectively (Chen et al., 2020).
2.6. Data analysis
As the fermentation went on, SLs was gradually accumulated in the broth
and once the O/S reached at a certain range, the SLs sedimentation was
occurred. The average sedimentation rate was calculated by formula (1).
By fitting the height of SLs sedimentation was directly proportional to
the concentration of SLs, formula (2) was obtained (Fig. 4A). The
sedimentation rate formula related to SLs concentration could be derived
in (3).
\(\overset{\overline{}}{V}=\frac{H_{1}-H_{2}}{2t}\) (1)
\(\frac{H_{2}}{H_{1}}=0.0031\times c-0.0001\) (2)
\(\overset{\overline{}}{V}=\frac{H_{1}-H_{1}(0.0031\times c-0.0001)}{2t}\)(3)
Where \(\overset{\overline{}}{V}\) is the average sedimentation rate of
SLs in cm/s, H1 is the height of broth in the 10 mL
sedimentation tube in cm, H2 is the height of SLs
settled in the 10 mL sedimentation tube in cm, t is settling time in s,
c is SLs concentration in g/L.
In this study, the sedimentation
mechanism and ultrasound assisted efficiency were analyzed by
the
Stokes
law as shown in (4) (Yang et al., 2015).
\(V=\frac{2r^{2}(\rho_{2}-\rho_{1})g}{9\mu}\) (4)
Where V is the sedimentation rate of SLs particles under ideal condition
in m/s, r is the radius of SLs particles in m, \(\rho_{1}\) is the
density of broth in kg/m3, \(\rho_{2}\) is the density
of SLs particles in kg/m3,g is the gravitational
acceleration in m/s2,\(\mu\) is the viscosity of
broth in Pa·s.
Due to the consideration of working volume change by feeding, all the
presented data in Figures and Tables were normalized to the initial
volume. All experiments were performed in triplicate.