Rezex roa organic acid h 8 150 7.8 mm column

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.

Prior to analysis, all samples were filtered through a PTFE filter (VWR, Leuven, Belgium). Naringenin and p-coumaric acid were quantified using a Waters Acquity UPLC H-Class system connected to an ACQUITY TUV-detector operating at 30 °C and 290 nm. A Kinetex^® 2.6 µm Polar C18 100 Å column (Phenomenex, Utrecht, The Netherlands) was used to separate metabolites using the following method, at a flow rate of 0.6 mL/min:

Time (min)	Eluent A: 0.1% TFA in water (%)	Eluent B: 100% acetonitrile (%)
0	90	10
0.5	75	25
5	75	25
7	30	70
8.5	30	70
10	90	10

Glycerol was quantified on a Shimadzu Prominence-I LC2030c Plus system connected to an RID-20A (Shimadzu) detector operating at 40 °C. A Rezex ROA-Organic Acid H + (8%) – 150 × 7,8 mm column (Phenomenex, Utrecht, The Netherlands) at 60 °C was used to separate metabolites using an isocratic method with a flow rate of 0.6 mL/min and 0.005 N H₂SO₄ in water as eluens.