After the overnight incubation samples were centrifuged at 13,000 x g for 5 min at room temperature.
The supernatants were collected and stored in new 2 ml tubes.
0.5 ml NaOH was again added to the remaining pellets and centrifuged at 13,000 x g for 5 min at room temperature.
The supernatants were combined and acidified with 300 µl of 32% HCl, for the ligno-thioglycolic acid complex precipitation.
Samples were incubated for 4 h at 4 °C with continuous shaking.
Thereafter, samples were centrifuged at 13,000 x g for 5 min at room temperature.
The supernatant was discarded and the lignin pellet was solved in 600 µl of 0.5 M NaOH.
The quantification of lignin was performed after appropriate dilution (if necessary) by measuring the absorbance at 340 nm. Calibration was performed using a standard curve made of lignin, alkali (Figure 1).