Rid 20a

The content of inulin, sucrose, glucose, and fructose in onion bulbs was determined by HPLC, which consisted of an autosampler (Shimadzu Nexera SIL-40CX3, Kyoto, Japan), a solvent delivery unit (Shimadzu Nexera LC-40DX3, Kyoto, Japan), a thermostatic column compartment (Shimadzu Nexera CTO-40C, Kyoto, Japan), and a refractive index detector (Shimadzu RID-20A, Kyoto, Japan). Sugar separation was achieved by injecting 10 µL of the sample into a calcium ion exchange column (300 × 8 mm, 9 µm particle size, Dr. Maisch ReproGel Ca, Ammerbuch, Germany) held at 80 °C using deionized water as the mobile phase (0.6 mL/min, isocratic elution). The identification and quantification of the investigated sugars were performed by comparing retention times and peak areas to analytical standards. The calibration curves were created by injecting serial dilutions of the investigated sugars (0.25, 0.50, 1.00, 2.50, 5.00, 7.50, and 10.00 g/L of inulin, sucrose, glucose, and fructose).

Short-chain fatty acid (SCFA) production was analyzed by high-performance liquid chromatography (HPLC). For this, 1.5 mL from stomach, ileum, or colon fermentation samples were taken and centrifuged at 10,000 x g for 10 min. Then, the supernatant was stored at −20°C until HPLC analysis. An SCFA standard curve from a stock solution (100 mM) containing lactate, formate, acetate, propionate, isobutyrate, and butyrate was prepared. Crotonate was used as the internal standard. Vials contained sample supernatants or standard curve and crotonate in a ratio 4:1 (v/v). SCFA production was measured with an LC 2030C HPLC (Shimadzu, Den Bosch, the Netherlands) equipped with a column Metacarb 67 h of 300 × 6.5 mm (Agilent, Amstelveen, the Netherlands) for the separation of organic acids. The injection volume was 20 μL. The column working temperature was 45°C, and the mobile phase was 0.01 N sulfuric acid at a flow of 1.0 mL/min. Metabolites were detected using a refractive index detector (RID-20A, Shimadzu, Den Bosch, The Netherlands).

Analysis of exo-metabolome was performed using filtered broth samples stored at -20°C until analysis. Organic acids and alcohols were analysed by HPLC (Shimadzu Prominence-I LC-2030 plus system) using a Rezex™ ROA-Organic Acids H⁺ (8%) 300 × 7.8 mm column (00H-0138-K0; Phenomenex) and a guard column (03B-0138-K0; Phenomenex). Twenty microlitres of the sample were injected using an auto-sampler and eluted isocratically with 0.5 mM H₂SO₄ at 0.6 mL/min for 30 min at 45°C. Compounds were detected by a refractive index detector (RID-20A; Shimadzu) and identified and quantified using relevant standards using the software LabSolution (Shimadzu). We note that cells produced 2R,3R-butanediol.

Organic fermentation products lactate, acetate and ethanol were analyzed on a Shimadzu 20A high-performance liquid chromatography (HPLC) system (Shimadzu). Metabolites were separated on a Rezex ROA-Organic Acid H + (8%) 150 × 7.8 mm column (Phenomenex) under isocratic temperature (65 °C) and flow (0.45 ml/min) conditions in 2.5 mM H₂SO₄ and then passed through a refractive index (RI) detector (Shimadzu RID-20A). Identification was performed by comparison of retention times with standards.
The yields were calculated assuming maximum formation of 1.67 mol or 2 mol of products (lactate + acetate + ethanol) from 1 mol of C5 or C6 sugar, respectively. The yields were calculated from the ratio between the total molar concentration of products (lactate + acetate + ethanol) formed upon growth on the respective substrates and the expected molar concentration of products at 100% utilization of C5 and C6 sugars in the substrate. Concentrations of glucose, xylose, galactose, arabinose and mannose in all substrates tested were determined according to standard procedure from NREL [41 ]. Mean values and standard deviations were obtained from three biological replicates.

The supernatant samples were centrifuged at 15,000 rpm for 10 min and filtered by 0.22 μm filters. The standards for the High-Performance Liquid Chromatography (HPLC) measurement of oxoglutarate, fumarate, malate, lactate, acetate, acetoin, glucose and ethanol were prepared with a set of concentration within 0–50 mM. All standards and samples were measured by HPLC using a Shimadzu instrument (LC-20AT, Prominence, Shimadzu) equipped with an Ion exclusion Rezex ROA-Organic Acid H+(8%) column (300 × 7.8 mm column, Phenomenex), along with a guard column (Phenomenex). Aqueous H₂SO₄ (5 mM) was used as mobile phase at a flow rate of 0.5 ml/min at 55 °C. A wavelength of 210 nm was used for calibration and analysis with the SPD-20 A UV/VIS detector, and a cell temperature of 40 °C was set for refractive index detector (RID 20 A, Shimadzu).