9. Triplicates must be performed for DCW determination per

sample.

Cell Viability Determination

1. Fermentation broth is diluted with pre-filtered 0.9% NaCl

solution to an approximate OD of 0.01–0.05 (see Note 6).

2. Stock solution of 0.5 mM DiBAC4(3) (bis-(1,3-dibutylbarbi-

turicacid-trimethineoxonol) is dissolved and diluted with

DMSO (dimethylsulfoxide) (see Note 7).

3. Diluted sample is fused with 1.5 μL of 0.5 mM DiBAC4(3)-

stock solution.

4. Additionally, the stain RH 414 (N-(3-triethylammoniumpro-

pyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)pyridinium

dibromide) could be added (1.5 μL of 2 mM stock solution) to

reduce background signals.

5. Incubate the sample for approximately 5 min in the dark.

6. Measure the sample with a suitable flow cytometry device (e.g.,

Partec, Cube 8, Sysmex, Germany).

7. Treat data with a suitable evaluation software (e.g., FACS

express 5.0, Sysmex, Germany); more details to this very

method can be found here [43].

3.3.2

Determination of

Metabolite Accumulation

Supernatant samples from sugar concentrations of feed and fer-

mentation broth are quantified via liquid chromatography.

1. Vortex fermentation broth after sampling to get homogenous

solution for 5–10 s.

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

3. Centrifuge the sample in a table centrifuge for 10 min at

11,180  g at 4
C.

4. Separate the supernatant from the pellet by decanting it.

5. Supernatant at this purification step contains of accumulated

sugars, required for quantification.

6. Filter supernatant samples at 0.2 μm prior to HPLC analysis.

7. Prepare an eluent at 0.1% H3PO4.

8. Prepare an anion exchange chromatography (e.g., Supelcogel-

AEX) for analysis at 30
C and a flow of 0.5 mL/min; Chro-

matogram recording should be carried out for 30 min.

9. Prepare standards at 1, 5, 10, 20, 30, 40, and 50 g/L of the

respective used carbon sources and accumulating metabolites

(see Note 8).

10. Chromatograms must be analyzed with a suitable processing

software (e.g., Chromeleon; Thermo Scientific, Waltham, MA,

USA).

234

Julian Kopp and Oliver Spadiut