(a)

Via an immersion pipe set to certain reactor volume,

which is connected via a tube to a peristaltic pump. For

continuous cultivation, pump settings given beneath need

to be adapted at all times: pump rate flow in < pump rate

flow out^.

(b)

Via gravimetric control of the reactor weight, which can

be monitored and controlled via a PI-controller in combi-

nation with a pneumatic valve. The valve opens once the

weight deviation exceeds a certain set-point and close

afterwards again.

3.2

Cultivation

Scheme

Cultivation scheme depicted in Fig. 2 represents a guidance to

cascaded continuous cultivation (see Note 2).

3.2.1

Preculture and

Batch Phase

1. All required media need to be sterilized at previously men-

tioned conditions (i.e., sterilization at 121
C or filtered at

0.2 μm in sterile environment).

2. Preculture is conducted as an overnight culture, i.e., 20 h, at

37
C, and 230 rpm.

3. In addition to Table 1, the pH for the preculture needs to be

set to 7.2 prior to sterilization with a 5 M NaOH stock

solution.

Fig. 1 Process overview of a continuous cascaded cultivation. Reactor 1 (i.e., stage 1) is used for biomass

formation. Biomass stream is transferred to reactor 2 (i.e., stage 2); recombinant protein production does only

take place in the second stage; 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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