Hyperrez carbohydrate h column

10 mL samples, collected from the bioreactor to determine biomass content, were centrifuged at 5500 g. The pellet was washed with distilled water, harvested by filtration and dried at 105 °C. The concentrations of glucose, polyols and citric acid in the supernatant from the samples were determined by HPLC using a HyperRez Carbohydrate H + Column (Thermo Scientific, Waltham, MA) coupled to a UV (λ = 210 nm) (Dionex, Sunnyvale, USA) and a refractive index detector (Shodex, Ogimachi, Japan).

Osmolality of some media was measured on the osmometer OS 3000 Marcel, Poland. The samples were diluted 10-fold before measurement. The concentrations of polyols and organic acids were determined by HPLC using a HyperRez Carbohydrate H+ Column (Thermo Scientific, Waltham, MA, USA) coupled to a UV (λ = 210 nm) (Dionex, Sunnyvale, CA, USA) and a refractive index detector (Shodex, Ogimachi, Japan). 0.25% trifluoroacetic acid was used as a mobile phase solvent. The samples were diluted 10-fold before the measurement. Data were analyzed with the Chromeleon program. Samples for biomass measurements (5 mL) were collected from shake-flask cultures and harvested by filtration on 0.45 µm pore size membranes. The biomass was determined gravimetrically after drying in a drier at 105 °C.

Samples (10 ml) from the cultures were centrifuged (10 min; 4°C; 5500 × g), harvested by filtration on 0.45-μm pore membranes and washed twice with distilled water. The biomass was determined gravimetrically after drying at 105°C. The concentration of polyols and citric acid in supernatant were determined with HPLC using a HyperRez Carbohydrate H⁺ Column (Thermo Scientific, Waltham, MA, United States) coupled to a UV (λ = 210 nm) (Dionex, Sunnyvale, CA, United States) and a refractive index (RI) detector (Shodex, Ogimachi, Japan). The column was eluted with 25 mM of trifluoroacetic acid (TFA) at 65°C and a flow rate of 0.6 ml min^-1.

Samples (10 mL) from the cultures were centrifuged (10 min; 4 °C; 5500×g), harvested by filtration on 0.45-μm pore membranes and washed twice with distilled water. The biomass was determined gravimetrically after drying at 105 °C. The concentrations of the metabolites were determined using HPLC equipped with a HyperRez Carbohydrate H⁺ Column (Thermo Scientific, Waltham, MA) coupled to a UV (λ = 210 nm) (Dionex, Sunnyvale, USA) and a refractive index (RI) detector (Shodex, Ogimachi, Japan). The column was eluted with 25 mM of trifluoroacetic acid (TFA) at 65 °C and a flow rate of 0.6 mL/min.

The samples obtained from the shake flask experiments were centrifuged for 15 min at 15,000 rpm. Supernatants were then transferred to new Eppendorf tubes and centrifuged again for 10 min at 15,000 rpm. To characterize the concentration of polyols (erythritol, arabitol and mannitol), glycerol and citric acid, the centrifuged samples were diluted 10 times and analyzed by HPLC analysis using a HyperRez Carbohydrate H + column (Thermo Scientific), coupled to a UV detector (λ = 210 nm) (Dionex, USA) and a refractive index detector (Shodex, Japan). Elution of the column was performed with 25 mM trifluoroacetic acid at 65 °C with a flow rate of 600 µL/min.

The concentrations of polyols and organic acids were determined by HPLC using a HyperRez Carbohydrate H+ Column (Thermo Scientific, Waltham, MA) coupled to a UV (λ = 210 nm) (Dionex, Sunnyvale, USA) and a refractive index detector (Shodex, Ogimachi, Japan). 0.25% trifluoroacetic acid was used as a mobile phase solvent. The samples were diluted 10-fold before the measurement. Data were analysed with the Chromeleon program.

During shake flask experiments, 1.5 mL samples were collected every 24 h and were centrifuged (5 min, 5000 rpm). The concentration of glycerol was determined in the supernatants by high-performance LC (HPLC, UltiMate 3000; Thermo Fisher Scientific, London, UK) equipped with the HyperRez Carbohydrate H + Column (Thermo Fisher Scientific) and a refractive index detector (Shodex, Ogimachi, Japan). Trifluoroacetic acetic acid at a concentration 25 mM was applied as an elution agent. Elution was carried out at a flow rate of 0.6 mL/min at 65 °C. The concentration of viscosinamide was measured from cell-free supernatant using HPLC (Shimadzu, Kyoto, Japan) equipped with the Hypersil GOLD column (5 µm, 4.6 × 150 mm). As the mobile phase, solvents A (0.1% trifluoroacetic acid) and B (0.1% trifluoroacetic acid in acetonitrile) were applied in the following order: (% A:B v/v): 0 min (50:50), 5 min (20:80), 9 min (10:90), 15 min (0:100), 21 min (0:100), 24 min (50:50), and 25 min (50:50). Samples were injected in a 10 µL volume on the Hypersil GOLD column (5 µm, 4.6 × 150 mm) and eluted for 25 min. Elution was performed at a flow rate of 0.5 mL/min and detection was conducted at a 210 nm wavelength.