Xylan

Conidia were inoculated at a concentration equal to 10⁶ conidia per ml in 3 ml Vogel's media (Vogel, 1956 ) with 2% wt/vol powdered Miscanthus giganteus (Energy Bioscience Institute, UC-Berkeley), Avicel PH 101 (Sigma-Aldrich, St. Louis, MO), beechwood xylan (Sigma-Aldrich), or pectin (Sigma-Aldrich) in a 24-well deep-well plate. The plate was sealed with Corning breathable sealing tape and incubated at 25°C in constant light and with shaking (200 rpm). Images were taken at 48 hr. Culture supernatants were diluted 200 times with 0.1 M NaOH before Dionex high-performance anion exchange chromatographic (HPAEC) analysis, as described below. N. crassa growth on xylan was also determined by measuring N. crassa biomass accumulation. N. crassa grown on xylan for 3 days was harvested by filtration over a Whatman glass microfiber filter (GF/F) on a Büchner funnel and washed with 50 ml water. Biomass was then collected from the filter, dried in a 70°C oven, and weighed.

To determine the lignocellulose-degrading capacity of strain CX11, enzymatic activity assays of fermentation broth were performed. The total cellulose-degrading activity, endo-1,4-beta-glucanase activity and xylanase activity were tested using Whatman No.1 filter paper, sodium carboxymethyl cellulose (CMC-Na) and xylan (from beechwood) as substrates, respectively. The generated reducing sugars were measured by the method of 3,5-dinitrosalicylic acid (DNS) as described previously (Miller 1959 (link)). p-Nitrophenyl-β-D-glucopyranoside (pNPG) was used as substrate to test the beta-glucosidase activity as reported before (Fang et al. 2014 (link)). The fermentation broth was boiled for 30 min to inactivate any possible enzymatic activities, and was used as a negative control. One unit (U) of enzyme activity was defined as the amount of enzyme required to release 1 μM of reducing sugar or pNP per minute, and was expressed as U mL⁻¹.