Lignin Degradation Compound Profiling

The Strain AORB19 was grown in 50 mL of culture medium with 0.1% Lignin, alkali from Sigma Aldrich as the carbon source and cultured at 27 °C on a shaker for 8 d. Every 48 h, a sample was sacrificed and centrifuged at 15,000 rpm for 10 min to remove the bacteria, and the culture supernatant was separated for further analysis. A control sample (culture media devoid of bacterial strain) was also prepared and subjected to the same treatment and analytical path. Phenolic compounds typical of Lignin degradation (i.e., phenol, o-catechol, 3-methylcatechol, guaiacol, syringol, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, vanillin, vanillic acid, syringaldehyde, syringic acid, acetovanillone, acetosyringone, ferulic acid) were qualitatively monitored in the supernatant using an Agilent 1260 Infinity II HPLC system equipped with a diode array detector (Agilent Technologies, Inc., Santa Clara, CA, USA). Separation of analytes was carried out with a Gemini® NX-C18 column (3 µm, 110 Å, 150 mm × 4.60 mm, Phenomenex, Torrance, CA, USA), at a temperature of 40 °C and a flow rate of 0.5 mL/min. A gradient of 5 to 95% MeOH in 0.044 N H₃PO₄ was used and detection was conducted at 280 nm. The injection volume was 100 μL.