2. According to determined maximum growth rate, turbidostat

experiments can be laid out, to determine washout of host cells.

(a)

This can be done in a single experiment increasing the

dilution rate in a stepwise approach.

(b)

Interval is adjusted according to μmax in 5–10% steps.

(c)

Each step needs to be held for 4–5 residence times until

biomass washout can be obtained.

3. Effect of dilution rate must be correlated to productivity: This

approach is confronted with a high experimental load where

effects of product stability need to be correlated to the dilution

rates.

(a)

Parallelization

increases

throughput

(i.e.,

multiple

induced reactors (Figs. 3 and 4)).

(b)

Design of experiment approaches can be used in case a

second factor influences productivity to reduce experi-

mental workload.

5.2

Substrate Uptake

Rate

1. In the first stage of cascaded continuous cultivation, μ can be

directly correlated to the specific substrate uptake rate.

2. In the second stage, substrate uptake rate can be either adjusted

via the ratio of inducer feed to the ratio of transferred cells or

the feed concentration; hence, Eq. 2 must be used for specific

substrate uptake rate calculation.

3. For experimental approaches on specific uptake rate determi-

nation, the following approach is utilized:

(a)

Inducer feed is set to lower values at first, whereas biomass

stream requires settings to a higher level to avoid washout

of host cells.

(b)

Desired ratios can be adjusted in a turibodstat-like

approach; i.e., start at 90% Biomass-stream (X1) and at

10% F2 or F3 of the overall dilution rate in stage 2.

4. When altering the overall dilution rate in stage 2 of cascaded

continuous cultivation, the approach shown in point 3 has to

be repeated until the desired specific substrate uptake rate is

adjusted. This is required as the average residence time influ-

ences substrate uptake rates.

5.3

Cultivation

Temperature

1. Effects of productivity in microbials is often correlated to

expression temperature. Temperature dependency thus needs

to be investigated.

2. As continuous processes suffer from a high experimental load,

screening for optimized production temperature is recommend

to be performed in fed-batch cultivations.

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

237