Lt200 system

Five mg of CWR were digested with 2.6 mL of 25% acetyl bromide in glacial acetic acid for 2 h at 50 °C using a HACH LT200 system. Samples were cooled on ice and transferred into 10 mL of 2 M NaOH plus 12 mL glacial acetic acid. The reaction tube was rinsed with glacial acetic acid and 1.75 mL of 0.5 M hydroxylammonium chloride was added. Each volume was adjusted to 30 mL with glacial acetic acid, centrifuged (3000 g, 15 min) and lignin content was measured spectrophotometrically (280 nm, extinction coefficient ε = 22.9 g^− 1 L cm^− 1).
Lignin was characterized following the nitrobenzene oxidation method previously described [69 (link), 78 (link)]. The products after nitrobenzene oxidation of ten mg of CWR were derivatized with Bis(trimethylsilyl)trifluoroacetamide and analysed by gas chromatography coupled to mass spectrometry (GC-MS). A HP-5MS column (30 m × 0.25 mm, 0.25 μm, Agilent) was used on a 7890B-5977A GC-MS system (Agilent). Salicylic acid-D4 was used as internal standard.

Lignin was characterised on 3 or 4 biological replicates using the nitrobenzene oxidation method [64 (link)]. 10 mg of CWR were digested with 2 mL of 2 M NaOH and 30 μL nitrobenzene at 165 °C for one hour (Hach LT200 system). After centrifugation, ca. 1500 μL of supernatant was collected and 10 μl of vanillin-D3 (Sigma-Aldrich) at 10 mg/mL in 1,4-dioxan were added as a surrogate standard. Nitrobenzene was removed by four washing steps with ethyl acetate (1 mL, vortexing/centrifugation cycle). The pH of the solution was adjusted to 2–3 by adding approximately 200 μL of 6 N HCl solution. The oxidation products were recovered by two successive extractions with 1 mL ethyl acetate (vortexing/centrifugation cycle) followed by cleaning with 500 μl of saturated NaCl solution and drying with Na₂SO₄. The GC-MS analysis was performed after trimethylsilylation, realized by addition of 50 μl of Bis(trimethylsilyl)trifluoroacetamide (BSTFA) to 50 μL of dried extract and derivatization at 60 °C for 30 min. Quantitative analyses were performed using a HP-5MS column (30 m × 0.25 mm, 0.25 μm, Agilent) installed in a 7890B-5977A GC-MS system (Agilent). Injection was done at 250 °C in splitless mode. The oven program started at 40 °C for 5 min, increased to 230 °C at 10 °C/min, then to 320 °C at 40 °C/min and was kept at 320 °C for 10 min. Salicylic acid-D4 was used as internal standard.