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. 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, corn steep liquor 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 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. Rapeseed oil
supplementation rate was 0.5 g/L/h
for the first 24 h, after which rapeseed oil concentration was
maintained at 2-15 g/L, according to rapeseed 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.