Allure organic acids column

We inoculated 0.5 mL of bacterial overnight cultures at ~1.5 of OD₆₀₀ into 25 mL of NBRIP with 0.5% of calcium phosphate in 125 mL of flask and incubated at 28 °C with shaking at 200 rpm for 4 days. The pH values were recorded with a pH meter. The bacterial cultures were centrifuged at 14,000 rpm for 10 min. The supernatant was filtered with a 0.2 µm filter column for HPLC injection. Gluconic acid was quantified using an Agilent 1100 HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a quaternary pump, autosampler, DAD detector and degasser.
The chromatographic separation of gluconic acid was achieved at ambient temperature using a 300 × 4.6 mm ID, 5 µm particle size Allure Organic Acids column (Restek, Bellefonte, PA, USA). Isocratic elution was carried out using a mobile phase of 100 mM phosphate buffer at 0.5 mL/min, and the retention time was 6.4 min with a 10 μL injection. A UV/Vis detector at 210 nm wavelength was used for detection. Pure gluconic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as a reference.

The analysis of δ¹³C values and concentrations of individual organic acids (citrate and malate) was performed by coupling an Isolink HPLC device to a DeltaV isotope ratio mass spectrometer (all Thermo-Fisher; Lehmann et al., 2015 (link), 2016b). Before analysis, the aqueous bulk organic acid fractions have to be passed through a 0.45 µm syringe filter (Infochroma, Zug, Switzerland). Organic acids were separated on a 4.6×300 mm Allure Organic Acids column (Restek, Bellefonte, PA, USA) at 8 °C using 100 mM KH₂PO₄ (pH 3) as a mobile phase with a flow rate of 500 µl min^–1. All organic compounds were oxidized to CO₂ at 99 °C using Na₂S₂O₈ under acidic conditions. The CO₂ was subsequently separated from the mobile phase and measured for its isotope ratio and concentration in the IRMS. Every 10 samples, a set of malate and citrate laboratory standards of different concentrations (10–180 ng C μl^–1) was analyzed. Offset corrections to EA-IRMS δ¹³C values and determination of concentrations were performed according to Rinne et al. (2012) (link). The applied method was shown to be free of isotope fractionation by analysis of standard materials with respect to the measurement precision, which was typically better than 0.4‰ (SD).

The final concentrations of the major organic acids, sugars, and ethanol were determined by HPLC, using a method according to Schneider et al. [59 ] and modified as described in [60 (link)]. An HPLC Agilent Technologies Series 1100, equipped with a binary pump, an autosampler, a multi-wavelength detector (MWD), and a refractive index detector (RID, Agilent Technologies, Steinheim, Germany) was employed. A column with a length of 250 mm, an inside diameter of 4.6 mm, and a particle size of 5 µm was used for the measurements (Allure Organic Acids Column, Restek, Bad Homburg v. d. Höhe, Germany). The MWD was set at a wavelength of 210 nm for the detection of organic acids and the RID was used for the detection of carbohydrates, organic acids, and ethanol. The eluent used was double distilled water with 0.5% ethanol and 0.0139% concentrated sulfuric acid. The flow rate was 0.6 mL/min at a temperature of 46 °C.