3.3

Sampling and

Analysis

Prior to induction, samples after batch are taken to determine

viable biomass growth and potential metabolite accumulation. An

additional sample after the continuous adaptation phase must be

taken to again determine viable biomass and metabolite accumula-

tion. Sampling frequency during induction is highly dependent on:

l

The doubling time of the host organism.

l

Expected deviations in productivity.

For E. coli BL21 (DE3), samples are taken once to twice a day.

In addition to previously stated analysis, product formation needs

to be monitored additionally after induction.

3.3.1

Biomass

Determination

Biomass is quantified via optical density (OD600) and gravimetri-

cally by weighing the dry cell weight (DCW) while flow cytometry

analysis (FCM) was used for the determination of cell death.

OD600 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. Vortex fermentation broth after sampling to get homogenous

solution for 5–10 s.

3. Determine OD600 using photometric cuvettes within the linear

range of the system (i.e., 0.2–0.8 [AU]).

4. In case the sample is not measured within linear range, dilute

sample after vortexing with deionized water; make sure to

never exceed 1:10 dilution steps due to accuracy.

5. Conduct at least duplicate measurements.

DCW Determination

1. Preheat 2-mL Safe-Lock Tubes (e.g., Eppendorf, Hamburg,

Germany) overnight at 105
C to free volatile compounds prior

to gravimetric determination.

2. Cool down tubes to room temperature, label reaction tubes,

and weigh the pre-tared amounts.

3. Vortex fermentation broth for 5–10 s after sampling to get

homogenous solution.

4. Pipette 1 mL in a pre-tared 2-mL reaction tube.

5. Centrifuge the sample in a small-scale centrifuge for 10 min at

11,180  g at 4
C.

6. Separate the supernatant from the pellet by decanting it (see

Note 5).

7. Immediately resuspend the pellet in 0.9% NaCl solution.

8. Centrifuge the dissolved pellet solution at the previously given

conditions (10 min at 11,180  g at 4
C).

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

233