Fed-batch experiments
An in-situ sterilizable 15 L bioreactor (BIOSTAT®B,
Germany)
equipped with pH and pO2 probes was used for fed-batch
experiments. One milliliter of glycerol stock bacteria
was inoculated into 100 mL LB medium
and cultured for 12 h at 37°C in a shaking incubator (200 rpm),
after which the pre-culture solution
was aseptically inoculated into 1 L LB medium and incubated under the
same conditions for 12 h. Next, 700 ml of seed cultures were inoculated
for 7-L fed-batch fermentation in the bioreactor. The value of oxygen
dissolved was set at 20%, which was cascade controlled by agitation
speed (300–1000 rpm) and air flow rate (3–20 L
min-1). The pH was set as 7.0 and automatically
controlled by adding 2 mol L-1 HCl or 2 mol
L-1 NaOH.
In the first fed-batch experiment (FB A), the temperature was adjusted
to 28°C at 3.5 h, while IPTG was added 30 min later as the
OD600 value reached about 18. A total of 600 mL feed
media was supplemented at 8 h with a flow rate of 30 mL
min-1. In the second fed-batch experiment (FB B), the
expression was induced at 5 h when the OD600 value had
reached 27, and the feed media was constantly fed after 14 h of
cultivation with a flow rate of 6 mL
min-1 for 1 h, then 3.6 mL min-1 for
2 h, and finally 2.4 mL min-1 for another hour. In the
third experiment (FB C), the expression was induced at 3 h when the
OD600 reached about 9 and the feed media was fed at 10
h. The feeding rate depended on the content of glycerol, which was
controlled at a concentration of 50 mmol L-1. Cell
growth was monitored at various times by measuring the
OD600 values.