Hence, the continuous adaptation phase only consists of feed-

ing in stage 1; however, no feed is added to stage 2, as this feed

contains of inducer.

2. For the cascaded continuous cultivations, stirred-tank reactors

are connected by transfer tubes coupled to peristaltic pumps

(e.g., Ismatec, Wertheim, Germany).

3. At the end of the batch phase, reactor temperature is adjusted

to desired values:

(a)

Stage 1: for biomass formation.

(b)

Stage 2: for recombinant protein production.

4. Continuous adaptation phase lasts for 4–5 residence times for

media exchange at least.

5. For screening of ideal process conditions, a higher amount of

induced bioreactors would reduce screening times; as biomass

Fig. 3 Process overview of the continuous cascaded cultivation. All lines in green are opened during

continuous adaptation phase, whereas black lines are closed. Reactor 1 ¼ stage 1 is used for biomass

formation. Biomass streams are separated equivalent into reactor 2 and 3 ¼ stage 2; Stage 2 is thus adapted

for continuous recombinant protein expression during the continuous adaptation phase. Di ¼ dilution rate

(1/h), Fi ¼ feed rate (L/h), Xi ¼ biomass flow (L/h), Pi ¼ product flow (L/h), Vi ¼ volume (L)

A Guideline to Set Up Cascaded Continuous Cultivation with E. coli Bl21 (DE3)

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