Hplc system

The content of caffeic and rosmarinic acid in leaves was determined as previously described (Kiferle et al., 2011 (link)). Briefly, the analyses were performed by using a Waters Alliance HPLC system equipped with a LiChrospher RP-18 column 250 mm × 4.6 mm, 5 μm (Phenomenex, United States), and the elution was carried out at a flowrate of 1.0 mL min^–1 at room temperature. Two mobile phases, A and B, were used: mobile phase A was acetonitrile, while mobile phase B was 0.1% phosphoric acid. The gradient used was 0–4 min, B 95%; 4–5 min, B 95–85%; 5–10 min, B 85–80%; 10–20 min, B 80–60%; 20–21 min, B 60–5%; 21–25 min, B 5%; 25–26 min, B 5–95%; and 26–30 min, B 95%. The volume injected was 20 μl, and the UV absorption was monitored at 325 nm.

Protocatechuic acid was analyzed using a Dionex HPLC system equipped with a UV/VIS detector and a Phenomenex kinetex 2.6 μm Biphenyl 100 A (50 × 2.1 mm) column for separation using a mobile phase consisting of 93% Solution A containing methanol: acetic acid: water (10:2:88) and 7% Solution B made of methanol: acetic acid: water (90:2:8), at a flow rate of 0.3 mL/min. Glucose, organic acids, and alcohols were analyzed by separation in a Jasco HPLC system equipped with RI detector using a BioRad Aminex HPX87H (Fast Acid) (100 × 7.8 mm) column. The mobile phase was 0.5 mM sulfuric acid used at a flow rate of 0.6 mL/min. The sample injection volume was 10 μL and all samples were filtered through a 0.2 μm filter, diluted 10 times in MQ water to a final volume of 1 mL prior to injecting into the column. The concentration of PCA and other metabolites formed was calculated as gram per liter (g/L) of the medium. Yield of PCA with respect to glucose (Y_P/S) was calculated as mol/mol. Biomass concentration was calculated as dry weight in cmol/L. The yield of PCA with respect to biomass (Y_P/B) was calculated as cmol/cmol. The productivity (g/L/h) was calculated by dividing maximum PCA concentration (g/L) by the time in hours from induction.