Securityguard guard cartridge

Ten milligrams of the biomass materials were suspended in 1 mL of 1 M NaOH and incubated for 1 h at 22°C. The mixture was centrifuged at 3,500 ×g for 5 min and the supernatant was filtered through a 0.22-μm pore-size filter hydrophobic membrane. Acetic acid was detected by HPLC featuring a Synergi Hydro-RP (Phenomenex, http://www.phenomenex.com) column equipped with SecurityGuard Guard Cartridge (Phenomenex) and the diode-array detector SPD-M20A (Shimadzu, http://www.shimadzu.com). To elute analytes, 20 mM K₃PO₄, pH 2.9, was used at the constant flow rate of 0.7 mL min⁻¹ at 22°C. Quantification was performed by integrating 210-nm peak area and comparing it with a standard curve generated using varying concentrations of acetic acid.

Extracellular concentrations of metabolites were determined on a Dionex Ultimate 3,000 HPLC system (Thermo Scientific, France) equipped with a RI detector (RID-10A, Shimadzu, Japan) and UV/Vis detector (SPD-20A, Shimadzu). The sample injection volume was 20 μL. Glucose and (D/L)-DHB were measured with the RI detector by using a cation-exchange column (Rezex RoA-organic acid H⁺ 8%) preceded by a SecurityGuard guard cartridge (Phenomenex, USA). The separation was performed at 80 °C with 0.5 mM H₂SO₄ at 0.5 mL min⁻¹ as mobile phase. PDO concentrations (from RI detector) were measured using an Aminex HPX-87H column protected by a Micro-Guard Cation H⁺ pre-column (BioRad, USA). The separation was performed at 35 °C with 1.25 mM H₂SO₄ at 0.5 mL min⁻¹. Amounts of D-DHB were estimated on the UV detector (at 254 nm) by using a Chirex 3126 column (Phenomenex, USA) with an aqueous mobile phase containing 2 mM CuSO₄ and 15% methanol. Flow rate was 1 mL/min, and the column was held at 22 °C. Concentrations of (L)-DHB were estimated by subtracting estimated amounts of the (D)-form to total (D/L)-DHB. All samples were centrifuged (2 min at 13,000 rpm) and syringe-filtered (0.2 µm), and the resulting supernatant stored at −20 °C before analysis. A standard calibration curve was obtained by injecting standards.