350 mM/L/h oxygen transfer rate obtained through
powerful fermentation agitation motor and 6-bladed
Rushton impellers dissolve 20% air saturation in the medium,
which was controlled by agitation at 500 rpm and
aeration rate 1 vvm. Unless otherwise stated, the pH of medium
was adjusted at 7.0 1 by the controlled addition of
NaOH (5 N) or 4 N HCl. The operations were controlled
and recorded by a digital control unit (DCU) in combination
with the software package. Samples of 10-20 mL were
withdrawn from the culture fluid for analytical purposes.
Bioreactor as a one-stage batch culture
In this experiment the fermentation vessel (bioreactor)
containing 9800 mL productive medium without
WFO was autoclaved at 121 °C for 40 min. WFO (1%) was
added after sterilization. The fermentation medium was inoculated
with 1% standard inoculum of the bacterial strain.
The standard inoculum was prepared in a conical flask
(250 mL) containing 100 mL of nutrient broth medium inoculated
with a loop of Ps. fluorescens S48 and incubated at
28-30 °C with shaking (300 rpm) for 24 h prior to inoculation
(5 x 108 cfu / mL).
The final working volume was 10 liter. Initial pH was
adjusted to 7 0.1 which was not controlled during the fermentation
period. Temperature, dissolved O2 and speed of
agitation were kept at 30 °C, 20% of air saturation and
500 rpm, respectively, during cultivation.
During fermentation, samples (10-20 mL) were withdrawn
from the culture (fermentation vessel) periodically.
The samples were centrifuged at 15000 xg for 4 min at 4 °C.
The sediment (biomass) was washed twice with distilled
water, and then dried at 70 °C to constant weight.
Bioreactor as a two-stage batch culture
The production of PHAs was carried out in two-stage
cultivation. In the first stage, two sterile conical flasks
(1000 mL) each containing 400 mL nutrient medium was
inoculated with 10 mL standard inoculum of the bacterial
strain, then incubated at 30 °C for 24 h on rotary shaker
(150 rpm) in order to get a luxurious growth. Then the culture
fluid was centrifuged at 15000 xg for 4 min at 4 °C and
the bacterial cells were collected and suspended in additional
sterile productive medium to inoculate the bioreactor
vessel to give a final working volume of 10 L sterile productive
medium. The cultivation conditions and microbiological
determinations were done as mentioned before.
Bioreactor as high-cell-density fed-batch culture
This experiment of fed-batch culture was constructed
to study the effect of washed high-cell-densities (0.64 g/L)
of Ps. fluorescens S48 on PHAs production. WFO was fed
continuously at 0.55 mL/L/h during the first 18 h of cultivation
(according to the obtained results from Gamal et al.
(2012) for the semi-scale production. Samples (10-20 mL)
were taken from the growing culture periodically under