2.5. Bacterial bioconversions in bioreactor
Bacterial bioconversions were carried out in a 1 L bioreactor
(New Brunswick Scientific Co., USA) with a working volume of
0.8 L. Temperature was set at 30 ◦C and the pH value was maintained
in the range of 6.7–6.9 using 5MNaOH and 10% (v/v) H2SO4.
The air flow rate was set constant at 1 vvm. The dissolved oxygen
(DO) was controlled at 25% of saturation through alteration of the
agitation speed. Hydrolysates were employed as the sole nutrient
feedstock combined with crude glycerol as carbon source at
an initial concentration of around 25 g L−1. During fed-batch fermentations,
concentrated crude glycerol (75%, v/v) was manually
added in the fermentation broth as feeding media. Foaming was
prevented via addition of an antifoam solution. Inoculation was
carried out using a 10% (v/v) 14 h pre-culture medium when the
bacteria were in the exponential growth phase.
Fermentation samples were centrifuged at 9000×g for 10 min
(Hettich Universal 320-R, Germany) and the supernatant was collected
for further analysis of free amino nitrogen (FAN), inorganic
phosphorus (IP) and glycerol. The sediment was washed with distilled
water and centrifuged prior to suspension in acetone in pre-weighed 14mL McCartney bottles for the determination of
residual cell mass (RCM) and PHB quantification.