5. All bioreactors need to be equipped with a dissolved oxygen

(dO2) probe to monitor dO2 in cultivation broth; e.g., a

fluorescence-dissolved oxygen electrode Visiferm DO (Hamil-

ton, Reno, NV, USA). Probes must be calibrated prior use. In

case dilution rates/residence times are adjusted via an immer-

sion tube and the fermentation broth volume, dO2 control is

only performed by adding oxygen as a stirrer-based control

would lead to deviations in the dilution rate.

6. All feeds and base/acids dosage bottles need to be placed on

data-recording scales.

7. Residual offgas from reactors needs to be quantified with

required offgas sensors (e.g., Blue Sens Gas analytics, Herten,

Germany).

(a)

Exhausting oxygen is quantified by zirconium oxide

sensors.

(b)

Exhausting carbon dioxide is quantified by near-infrared

spectra analysis.

2.4

Required

Equipment for Process

Analysis

2.4.1

Biomass

Determination

1. Any photometric device/plate reader quantifying the absorp-

tion in liquid broth can be used, e.g., a Genesys 20 photometer

(Thermo Scientific, Waltham, MA, USA).

2. Cuvettes for OD600 determination.

3. 2-mL Eppendorf Safe-Lock Tubes (Eppendorf, Hamburg,

Germany).

4. Oven at 105
C.

5. Small-scale centrifuge capable of cooling and spinning down

2-mL safe lock tubes (Eppendorf, Hamburg, Germany).

6. Filtered 0.9% NaCl solution for washing steps.

2.4.2

Viable Biomass

Determination

1. Flow cytometry device with integrated 488 nm laser and detec-

tor cell (e.g., Partec, Sysmex Germany).

2. Fluorescent stains for live–dead determination, e.g., DiBAC4

(3) (bis-(1,3-dibutylbarbituricacid-trimethineoxonol).

3. Filtered 0.9% NaCl solution for dilution steps.

2.4.3

Metabolite

Determination

1. Liquid chromatography device including refractory index

(RI) detector (e.g., Dionex Ultimate 3000 SD, Thermo Scien-

tific, Waltham, MA, USA); the device must include:

(a)

Pump module.

(b)

Auto-sampler module.

(c)

Oven module.

(d)

Detector module.

(e)

RI detector module.

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

227