4

Notes

1. If glycerol is exchanged by glucose, carbon source needs to be

sterilized separately in split volumes. MgSO4 · 7H2O, Fe(III)

citrate, and EDTA were sterilized at 121
C for 20 min. All

other chemicals were filtered at 0.2 μm under sterile conditions

in pre-sterilized bottles.

2. Please note that process times are highly dependent on the host

organism and are given in this case for E. coli BL21(DE3).

3. Initial substrate concentrations beyond 50 g/L might lead to

high metabolite accumulation (i.e., acetate) and must be

avoided.

4. Even though productivity might be constant in stage 2, the

offgas signal might deviate due to subpopulation diversifica-

tion, potentially resulting in different sugar uptake rates; still

offgas signals in the seed reactor should be constant at all time.

5. Supernatant at this stage contains accumulated sugars/proteins

and requires storage at 20
C for further analysis.

6. Dilutions with deionized water might lead to deviations in

quantification (due to osmotic pressure and subsequent cell

lysis); dilution steps should thus not exceed 1:50 dilutions.

7. Flow cytometer stain is light sensitive and thus requires ade-

quate storage. DiBAC4(3) can only penetrate cells, if the cell

wall is damaged. Due to an increase in the fluorescent gain, a

differentiation between viable and non-viable cells can be

obtained.

8. Each metabolite can result in an altering RI-fingerprint; hence,

calibration is only valid with respective standard substance.

5

General Notes on Process Intensification for Cascaded Continuous Cultivation

For E. coli BL21 (DE3), a careful optimization strategy has to be

carried out in microbial continuous cultivation regarding:

(a)

Dilution rates.

(b)

Substrate uptake rate.

(c)

Cultivation temperature.

5.1

Dilution Rate

1. Maximum growth rate is determined in a batch culture.

(a)

A fit of residual carbon dioxide signals can be performed.

(b)

Frequent sampling and determination of biomass growth

and carbon reduction has to be done.

(c)

A 96-well plate reader screening optical density at con-

trolled environments (e.g., Tecan, M€annedorf, Switzer-

land) can be used to determine μmax.

236

Julian Kopp and Oliver Spadiut