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.