Cto 20a

The GCPs (3 × 10⁶ cells) were suspended in 0.4 M mannitol, 10 mM KCl, 0.5 mM CaCl₂, 0.5 mM MgCl₂ and 5 mM MES-Tris buffer (pH 6.0) and illuminated with white light (80 μmol m^-2 s^-1 for 1 h) as described elsewhere [22 (link)] before isolation of the ions. The GCPs were ground into a fine powder, suspended in distilled water and boiled for 5 min. The suspensions were applied to Microcon YM-10 centrifugal filters (Millipore), and the organic/inorganic ions were measured in the filtrate.
The measurement of organic and inorganic ions was performed on an HPLC (Prominence, Shimadzu). The chromatographic system consisted of a Shimadzu Model system controller (SLC-10AVP), conductivity detector (CDD-10ADVP), and column oven (CTO-20A). K^- and Na⁺ were separated using a LC-10ADVP pump, a column Shim-pack IC-C4 (150 mm × 4.6 mm) and a guard column Shim-pack IC-GC4 (10 mm × 4.6 mm) with a mobile phase (3.2 mM Bis-Tris, 8 mM p-hydroxybenzoic acid, 50 mM Boric acid) and a flow rate of 1.0 ml min^-1 at 40°C. Cl^- and Malate^2- were separated with a LC-20AD pump, a column Shim-pack IC-A3 (150 mm × 4.6 mm) and guard column Shim-pack IC-GA3 (10 mm × 4.6 mm) with a mobile phase (3/4 dilution of 3.2 mM Bis-Tris, 8 mM p-hydroxybenzoic acid, 50 mM boric acid) and a flow rate of 1.2 ml min^-1 at 40°C. Statistical analyses were performed using the Student's t test.

High-performance liquid chromatograph system–Shimadzu LC-20 Nexera XR, solvent supply system—LC-20AD, degasser—DGU-20A5R, autosampler—SIL-20A, detector—SPD-M20A (Diode array detector), column thermostat—CTO-20A from (Shimadzu Europa GmbH, Duisburg, Germany); column—ZORBAX Eclipse Plus, C18, 150 × 4.6 mm, 3.5 µm, from Agilent (Santa Clara, CA, USA).
The mobile phase, consisting of a mixture of water phase (containing 67 mmol·L⁻¹ potassium dihydrogen phosphate) and acetonitrile in ratio 75:25 (v/v, each liter of mobile phase containing 1.7 g tetramethylammonium sulfate) was delivered isocratically at a flow rate of 1.0 mL·min⁻¹. Following its preparation, the mobile phase was filtered under vacuum through a 0.45 μm membrane filter and ultrasonically degassed prior to use. The chromatographic separation was performed on a ZORBAX Eclipse Plus, C18, 150 × 4.6 mm, 3.5 µm, from Agilent (Santa Clara, CA, USA). The column temperature was maintained constantly at 40 °C using a thermostatically controlled column oven. UV-Vis spectrophotometric detection was performed at 237 nm wavelength. The chromatographic running time for each analysis was 5.0 min.

Quantification of organic acids by Aminex HPX-87H column (300 × 7.8 mm, Bio-Rad, Hercules, CA, USA) was performed as described previously [34 ] with some modifications. The mobile phase was 3 mM sulfuric acid aqueous solution. Sample (20 µL) was separated on a column set at 42 °C with a flow rate of 0.6 mL/min; and the absorbance at 215 nm was monitored by an SPD-M20A detector (Shimadzu, Japan). The HPLC system consisted of two pumps (LC-20 AT, Shimadzu, Kyoto, Japan), a degasser (DGU-20A, Shimadzu), an autosampler (SIL-20 A, Shimadzu) and a column oven (CTO-20A, Shimadzu). Chromatograms were evaluated with the Clarity software package (LabSolutions, Shimadzu). This method was termed as HPLC method 1.