3.8. T. reesei Strain Cultivation Induced by FeCl3 and Enzyme-Activity Determination

The T. reesei Rut-C30 strain (CICC 40348, from China Center of Industrial Culture Collection) was grown on potato-dextrose agar (PDA) at 30 °C for 7 d, and the spores were harvested with double distilled water and counted on the hemocytometer. The germination rate of spores was accurately examined for appropriate incubation time prior to micro-fluidic analysis and sorting. The spores were collected and adjusted to a density of 1 × 10⁷ spores/mL. A spore suspension of about 500 μL was added into 30 mL of liquid cellulase-inducing medium, incubated at 30 °C under shaking at 200 rpm for 7 d. The liquid cellulase-inducing medium was prepared as previously described [37]. About 0.6 g of biomass powder of corn stalk without soluble sugars was added into the liquid cellulase-inducing medium as carbon source and induced by FeCl₃ at various concentrations (0%, 0.01%, 0.05%, and 1%; w/v).

Filter-paper activity (FPA) of crude cellulase solutions secreted by T. reesei was determined as described previously [37]. Exoglucanase (CBH), endoglucanase (EG), β-glucosidase (BG), and xylanase activities of crude cellulase solutions secreted by T. reesei were examined in vitro by using carboxymethylcellulose sodium (CMC-Na), 4-Nitrophenyl β-D-cellobioside (pNPC), salicin, and beechwood xylan (from China National Pharmaceutical Group Co., Ltd., Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China) substrates dissolved with sodium-citrate buffer (0.05 M, pH 4.8) as previously described [37,62]. All assays were completed in independent triplicates.

Total proteins secreted by T. reesei were estimated as previously described [37]. The SDS-PAGE was run for profiling all proteins secreted by T. reesei using stain-free precast gels (Zoman Biotechnology Co., Ltd., Beijing, China), according to the manufacturer’s instructions. All crude enzymes secreted by T. reesei were analyzed by LC–MS/MS (Jingjie PTM BioLab Co., Ltd., Hangzhou, China; Orbitrap Elite LC–MS/MS, Thermo, Waltham, MA, USA), as described in [37]. Liquid-chromatography–MS/MS-analysis data were identified by searching the T. reesei Rut-C30 protein-sequence databases downloaded from Uniprot (https://www.uniprot.org/), accessed on 30 April 2022.