Standard microbial expression systems, the bacterium Escherichia coli and the yeast Hansenula polymorpha, were used to evaluate the improved measurement device. The microorganisms were provided by several cooperation partners. The strain E. coli BL21-Pet 28A ytvAC62A, expressing a flavin mononucleotide (FMN)-based fluorescent protein (FbFP) [22 (link)], was kindly delivered by Thorsten Eggert from evocatal GmbH, Germany (FbFPs reporter proteins are commercialised under the name evoglow®,
The E. coli experiments were carried out with three different types of bacterial culture media: complex medium Luria Bertani (LB), Terrific Broth (TB) and the synthetic medium for E. coli fedbatch fermentations reported by Wilms et al. (WR) [24 (link)]. These media had the following compositions: LB medium: 10 g/L tryptone (Difco from Becton Dickinson, USA), 5 g/L yeast extract (Roth, Germany) and 5 g/L NaCl, pH~6.7 without adjustment; TB medium: 5 g/L glycerol (Merck, Germany), 12 g/L tryptone, 24 g/L yeast extract, 12.54 g/L K2HPO4, 2.31 g/L KH2PO4, pH~7.2 without adjustment; WR medium: 2.0 g/L Na2SO4, 2.68 g/L (NH4)2SO4, 0.5 g/L NH4Cl, 14.6 g/L K2HPO4, 4.0 g/L Na2HPO4 × 2 H2O, 1.0 g/L (NH4)2-H-citrate, 0.5 g/L MgSO4 × 7 H2O, 0.01 g/L thiamine, 3 ml/L trace element solution (TES), 20 g/L glucose or glycerol, pH was adjusted to 7.2 with 1 M NaOH. TES contains: 0.5 g/L CaCl2, 0.18 g/L ZnSO4 × 7 H2O, 0.1 g/L MnSO4 × H2O, 10.05 g/L Na2-EDTA, 8.35 g/L FeCl3, 0.16 g/L CuSO4, × 5 H2O, and 0.18 g/L CoCl2 × 6 H2O. The E. coli cultures were induced with 0.5 mM isopropyl-β-D-thiogalactopyranosid (IPTG, Biomol, Germany).
To study H. polymorpha with different media, the following media were applied: YPG, YPD, YNB-G, YNB-D (buffered and unbuffered). YP medium contained: 20 g/L peptone (Difco from Becton Dickinson, USA), 10 g/L yeast extract (Roth, Germany) and 10 g/L glycerol (YPG) or 20 g/L glucose (YPD), pH~7.6 without adjustment. YNB medium contained: 5 g/L (NH4)2SO4 and 1.7 g/L YNB without ammonium sulfate and amino acids (Difco from Becton Dickinson, USA) and 10 g/L glycerol (YNB-G) or 20 g/L glucose (YNB-D). After all medium components were dissolved, the pH was adjusted to 6.0 with 1 M NaOH. The buffered YNB medium was supplemented with Na2HPO4/NaH2PO4 buffer in a concentration of 0.1 M to maintain the pH at about 6 (starting value pH0 = 6.0) during the batch fermentation. In the experiments with different glycerol concentrations, the glycerol concentration in the YNB medium was simply varied during media preparation, whereas the other components remained constant. All the applied chemicals were of analytical grade and were delivered by Fluka (Neu-Ulm, Germany), unless specified otherwise.
The E. coli experiments were carried out with three different types of bacterial culture media: complex medium Luria Bertani (LB), Terrific Broth (TB) and the synthetic medium for E. coli fedbatch fermentations reported by Wilms et al. (WR) [24 (link)]. These media had the following compositions: LB medium: 10 g/L tryptone (Difco from Becton Dickinson, USA), 5 g/L yeast extract (Roth, Germany) and 5 g/L NaCl, pH~6.7 without adjustment; TB medium: 5 g/L glycerol (Merck, Germany), 12 g/L tryptone, 24 g/L yeast extract, 12.54 g/L K2HPO4, 2.31 g/L KH2PO4, pH~7.2 without adjustment; WR medium: 2.0 g/L Na2SO4, 2.68 g/L (NH4)2SO4, 0.5 g/L NH4Cl, 14.6 g/L K2HPO4, 4.0 g/L Na2HPO4 × 2 H2O, 1.0 g/L (NH4)2-H-citrate, 0.5 g/L MgSO4 × 7 H2O, 0.01 g/L thiamine, 3 ml/L trace element solution (TES), 20 g/L glucose or glycerol, pH was adjusted to 7.2 with 1 M NaOH. TES contains: 0.5 g/L CaCl2, 0.18 g/L ZnSO4 × 7 H2O, 0.1 g/L MnSO4 × H2O, 10.05 g/L Na2-EDTA, 8.35 g/L FeCl3, 0.16 g/L CuSO4, × 5 H2O, and 0.18 g/L CoCl2 × 6 H2O. The E. coli cultures were induced with 0.5 mM isopropyl-β-D-thiogalactopyranosid (IPTG, Biomol, Germany).
To study H. polymorpha with different media, the following media were applied: YPG, YPD, YNB-G, YNB-D (buffered and unbuffered). YP medium contained: 20 g/L peptone (Difco from Becton Dickinson, USA), 10 g/L yeast extract (Roth, Germany) and 10 g/L glycerol (YPG) or 20 g/L glucose (YPD), pH~7.6 without adjustment. YNB medium contained: 5 g/L (NH4)2SO4 and 1.7 g/L YNB without ammonium sulfate and amino acids (Difco from Becton Dickinson, USA) and 10 g/L glycerol (YNB-G) or 20 g/L glucose (YNB-D). After all medium components were dissolved, the pH was adjusted to 6.0 with 1 M NaOH. The buffered YNB medium was supplemented with Na2HPO4/NaH2PO4 buffer in a concentration of 0.1 M to maintain the pH at about 6 (starting value pH0 = 6.0) during the batch fermentation. In the experiments with different glycerol concentrations, the glycerol concentration in the YNB medium was simply varied during media preparation, whereas the other components remained constant. All the applied chemicals were of analytical grade and were delivered by Fluka (Neu-Ulm, Germany), unless specified otherwise.
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