Growth was monitored by measuring turbidity at 600 nm (OD600), cell counts, and biomass dry weight (DW). Cells were counted in a Bürker chamber; the DW was determined with a thermo-balance (MB 64 M, VWR, Radnor, PA, USA) (20).
Arabitol and residual glycerol in culture supernatants—clarified by centrifugation (10,000 rpm for 5 min at 4 °C) and filtration at 0.22 μm—were analyzed in HPLC with refractive index detector (1200 System, Agilent Technologies, Waldbronn, Germany). Isocratic elution was carried out at 60 °C with 0.8 mL/min of 5 mM H2SO4 through an ion exclusion column (Aminex HPX-87 H, Bio-Rad, Hercules, CA, USA) [27 (link)]. 1 H- spectra were recorded at 298 K on Bruker FT-NMR Advance 400 (400.13 MHz). Chemical shift values are given in ppm relative to TMS and were determined by taking as reference the isotopic impurity signals DMSO-d6 (2.50 ppm). Prior to NMR analysis, the supernatant of the improved fed-batch cultures was lyophilized overnight with a Alpha 1–2 LD Laboratory (Christ, Germany). Polarimetric analysis was carried out using a Polax-2 L polarimeter (Atago, Japan).
Arabitol and residual glycerol in culture supernatants—clarified by centrifugation (10,000 rpm for 5 min at 4 °C) and filtration at 0.22 μm—were analyzed in HPLC with refractive index detector (1200 System, Agilent Technologies, Waldbronn, Germany). Isocratic elution was carried out at 60 °C with 0.8 mL/min of 5 mM H2SO4 through an ion exclusion column (Aminex HPX-87 H, Bio-Rad, Hercules, CA, USA) [27 (link)]. 1 H- spectra were recorded at 298 K on Bruker FT-NMR Advance 400 (400.13 MHz). Chemical shift values are given in ppm relative to TMS and were determined by taking as reference the isotopic impurity signals DMSO-d6 (2.50 ppm). Prior to NMR analysis, the supernatant of the improved fed-batch cultures was lyophilized overnight with a Alpha 1–2 LD Laboratory (Christ, Germany). Polarimetric analysis was carried out using a Polax-2 L polarimeter (Atago, Japan).
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