SCFA Quantification by HPLC

Chromatographic analyses for SCFA detection were made as described previously (Adamberg et al. 2020 (link)): Culture supernatants were filtered using AmiconR Ultra 10 K Centrifugal Filter Devices, cut-off at 3 kDa according to the manufacturer’s instructions (Millipore, United States). The concentrations of organic acids (succinate, lactate, formate, acetate, propionate, butyrate) and free sugars (mono-, di-, and trisaccharides) were determined by high performance liquid chromatography (HPLC, Alliance 2795 system, Waters, Milford, MA, United States), using Bio-Rad HPX-87H column (Hercules, CA, USA) with an isocratic elution of 0.005 M H₂SO₄ at a flow rate of 0.5 mL/min and at 35 °C. Refractive index (RI) (model 2414; Waters, USA) and UV (210 nm; model 2487; Waters, USA) detectors were used for quantification of the substances. The detection limit for the HPLC method was 0.1 mm.