Whatman grade 1 filters

T. reesei and A. niger spores were harvested from fresh potato dextrose agar (PDA) plates by adding 1 mL of sterile distilled water. The spore suspensions were inoculated in triplicate to a final concentration of 1 × 10⁶ spores per 30 mL of BCM (pH5.5) in a 250-mL Erlenmeyer flask containing 1% fructose (w/v) as the sole carbon source. T. reesei and A. niger spores were grown at 29°C and 30°C, respectively, for 24 hours (A. niger) on a rotary shaker with agitation at 200 rpm. T. reesei was also grown on a rotary shaker with agitation of 200 rpm, but it was grown for 48 hours to achieve an initial mycelial mass similar to that of A. niger. After, mycelia were removed by filtration through Whatman grade 1 filters (GE Healthcare), and they were washed with sterile water and transferred to 30 mL of fresh BCM media (without yeast extract) containing 0.5% of SEB or SC (w/v) as the sole carbon source for 6, 12 or 24 hours. T. reesei cultures were grown in a controlled environmental growth chamber under constant illumination with white light.
The mycelia and biomass used as carbon sources were harvested by filtration, washed thoroughly with sterile water and quickly frozen in liquid nitrogen for further cell wall monosaccharide composition analyses. The supernatant was stored at -20°C for enzymatic, soluble supernatant sugar and mass spectrometry analyses.

A. fumigatus AF293, gently donated by the Prof. Dr. Sérgio Akira Uyemura (University of São Paulo, BR), was grown on YAG medium (2% (w/v) dextrose, 0.5% (w/v) yeast extract, 0.1% (v/v) trace elements and 1.8% (w/v) agar) at 37 °C for two days. Spores were harvested and inoculated to a final concentration of 1 × 10⁸ per 50 mL of YNB culture with 1% (w/v) fructose as the carbon source at 37 °C for 16 h (h) in a rotary shaker with agitation at 200 rpm. Afterward, the mycelia were transferred to 1% (w/v) SEB (47.5% cellulose; 9.0% hemicellulose and 34.3% lignin) or 1% (w/v) fructose as the carbon source for 3, 6, 12, 18, 24, 48 and 72 h. Fructose was used as a control in all experimental conditions [26 (link)]. Mycelia were harvested by filtration through Whatman grade 1 filters (GE Healthcare, Grandview Blvd. Waukesha, WI, USA), washed once with sterile cool water and kept at − 80 °C until RNA extraction. Supernatants were collected to measure enzymatic activity and determine the secretome. All the experiments described below were performed in three biological replicates.