Alkali lignin

The following reagents were purchased from commercial suppliers: aluminum isopropoxide (Fuchen Co., Ltd., Tianjin, China, 98.5%), sodium hydroxide (Pengkun Co., Ltd., Tianjin, China, 96.0%), tetraethyl orthosilicate (Fuchen Co., Ltd., Tianjin, China, 98.5%), tetrapropylammonium hydroxide (Siyoupu Co., Ltd., Hefei, China, 1 mol/L), niobium oxalate (Haoxuan Co., Ltd., Guangzhou, China, 95.0%), ammonia chloride (Nankai Co., Ltd., Tianjin, China, 99.5%), and alkali lignin (Merck KGaA, Darmstadt, Germany, 99.5%.

Fisherbrand™ Qualitative Grade Plain Filter Paper P4 (~98 g/m²) and P8 (~68 g/m²) was purchased from Fisher Scientific (Ottawa, ON, Canada) and used as base paper for this study. Alkali lignin (AL) was purchased from Sigma Aldrich (St. Louis, MO, USA). Nanofibrillated cellulose (NFC) was obtained from University of Toronto (Toronto, ON, Canada). All the other chemicals were purchased from Sigma Aldrich and used as received without further purification.

Isolated bacterial colonies were transferred to minimal media (6 g/L NaCl, 1 g/L (NH₄)₂SO₄, 0.5 g/L KH₂PO₄, 0.5 g/L K₂HPO₄, 0.1 g/L MgSO₄, 0.1 g/L CaCl₂, 2 g/L MES, 15 g/L agar, pH 5.75 using NaOH) supplemented with 1 g/L of either alkali lignin, colloidal microcrystalline cellulose, or birchwood xylan (all from Sigma-Aldrich Corp.) as sole carbon sources. Plates were incubated at 37 °C for 4 days, and bacteria were transferred to fresh plates in order to confirm the results.
Cellulose degradation was detected by transferring colonies to minimal medium containing 1 % w/v carboxymethylcellulose and 0.1 w/v glucose. After 48 h, the plate was flooded with 5 mL of 0.1 % w/v Congo red (Ricca Chemical Co., Arlington, TX, USA) and stained for 15 min at room temperature. After removing the dye, the plate was washed twice with 5 mL 1 M NaCl for 15 min each, then air dried and photographed.
Ligninolytic enzyme production was detected by transferring colonies to minimal medium containing 25 mg/L methylene blue at 28 °C for 72 h.

For DR5B decolorization, 10 mL of the pre-cultured I. lacteus CD2 was inoculated into 100 mL modified Kirk medium supplied with 100 mg/L DR5B (purchased from Colorfran S.A., Mexico) and cultivated at 30 °C with the shaking speed at 150 rpm for 5 days. To understand the effect of alkali lignin (purchased from Sigma-Aldrich, USA) on DR5B decolorization by I. lacteus CD2, 30 mg/L of lignin was added to the aforementioned dye decolorization medium. Meanwhile, additional two experiments were set up as controls: 1) I. lacteus CD2 grown on modified Kirk medium; 2) I. lacteus CD2 grown on modified Kirk medium supplied with 30 mg/L of lignin. The decolorization rate of DR5B was measured by UV/vis spectrophotometer at the wavelength 510 nm.

Beechwood xylan, 3, 5-dinitrosalicylic acid (DNSA), Congo red, d-xylose, alkali lignin and phenolics were purchased from Sigma (St. Louis, MO, USA). DEAE-Cellulose and sorbitol were from Merck Bioscience. All other chemicals and solvents used in this work were of analytical grade and obtained from S. D. Fine Chem. Ltd., Mumbai, India. Microbiological culture media and media ingredients were obtained from HiMedia (Mumbai, India). The wheat bran (Triticum aestivum) was obtained from the local market in Lucknow (U. P.), India.