Quantification of Vanillin Production

For small-scale production of vanillin, experiments were carried out using 100-ml shake-flasks. 1% fresh overnight culture was inoculated into shake-flasks containing 10 ml SC medium supplemented with 20 μM copper sulfate. The cultures were incubated at 30 °C and 250 rpm for vanillin productions. For gas chromatography-flame ionization detector (GC-FID) analysis of vanillin, 100 μl of supernatant was extraction with 900 μl ethyl acetate before subjected to GC-FID analysis. 1 μl of diluted sample was injected into GC-2030 system equipped with an Rtx-5 column (30 m × 250 μm × 0.25 μm thickness). Nitrogen (ultra-purity) was used as carrier gas at a flow rate 1.0 ml min⁻¹. GC oven temperature was initially held at 40 °C for 2 min, increased to 45 °C with a gradient of 5 °C min⁻¹ and held for 4 min. And then it was increased to 230 °C with a gradient 15 °C min⁻¹.
For high-performance liquid chromatography (HPLC) analysis of vanillin, Shimadzu Prominence LC-20A system (Shimadzu, Japan) equipped with a reversed phase C18 column (150 × 4.6 mm, 2.7 μm) and a photodiode array detector was used. The samples were centrifuged and filtered through a 0.2-μm syringe filter before injected to the HPLC system. The mobile phase comprises solvent A (ddH₂O with 0.1% trifluoroacetic acid) and solvent B (acetonitrile with 0.1% trifluoroacetic acid). The following gradient elution was used: 0 min, 95% solvent A + 5% solvent B; 8 min, 20% solvent A + 80% solvent B; 10 min, 80% solvent A + 20% solvent B; 11 min, 95% solvent A + 5% solvent B. The flow rate was set at 1 ml min⁻¹. The levels of vanillin and other aromatic compounds were monitored at the absorbance of 275 nm.