Microorganisms and inoculum
Trichoderma reesei (BNCC339931) was obtained from the BeNA Culture Collection. Trichoderma harzianum (CICC41290) was obtained from the China Center of Industrial Culture Collection. Aspergillus niger (ATCC16404) was purchased from the American Type Culture Collection. These fungi were stored on PDA plates at 4 °C and transformed to fresh PDA medium monthly and cultured at 25 °C for 5d. After growth on the plate, the spores were washed with 3 mL distilled water to obtain spore suspension. The inoculum was prepared by culturing the fungi under submerged fermentation in 300 mL Erlenmeyer ﬂasks, the seed medium was prepared as following (g/L): glucose 10, peptone 1, Tween-80 0.002, (NH4)2SO4 1.4, KH2PO4 2, urea 0.3, MgSO4 0.3, CaCl2 0.4, ZnSO4 0.0014, MnSO4 0.0016, FeSO4 0.005, citric acid buffer 500 mL/L .
Cellulase production by submerged fermentation
The single and combination carbon source (ramie, wheat bran or avicel) were studied to evaluate their effects on enzymes production from A. niger. The concentration of carbon sources was listed as following: ramie (80 g/L), wheat bran (80 g/L), avicel (80 g/L). The fermentation broth (75 mL) containing (NH4)2SO4 (4.0 g/L), KH2PO4 (2.0 g/L), MgSO4.7H2O (0.3 g/L), tryptone (3.0 g/L) and yeast extract (0.5 g/L).
The carbon source including ramie, wheat bran, lactose or avicel, and nitrogen source including ammonium sulphate, ammonium chloride tryptone or yeast extract for T. reesei and T. harzianum submerged fermentation (SmF) was evaluated, their concentrations were set as follows: ramie (10, 20 and 30 g/L), wheat bran (10, 20 and 30 g/L), lactose (10, 20 and 30 g/L), avicel (10, 20 and 30 g/L), ammonium sulphate(4, 6, 8 and 10 g/L), ammonium chloride (4, 6, 8 and 10 g/L), tryptone (1, 2, 3 and 4g/L) and yeast extract (1, 2, 3 and 4 g/L). The fermentations of T. reesei and T. harzianum were carried out in 250 mL Erlenmeyer ﬂasks with 50 mL working volume,the culture media contains(g/L): KH2PO4, 2.0; MgSO4, 0.3 and Tween 80, 0.2, MnSO4 0.0016, ZnSO4 0.0014 mg and FeSO4 0.005.
All mediums were autoclaved at 120 °C for 20 min, then 10% of inoculum inoculated after the temperature was decreased to room temperature. The mixtures were cultured at 30 °C, the extraction of enzymes was performed after fermentation of T. reesei and T. harzianum for 36 h, and fermentation of A. niger for 72 h. The supernatant was collected by ﬁltering through nylon cloth followed by centrifugation at 10,000 rpm for 10min. All experiments were performed in triplicate.
Optimization of pH, temperature and fermentation time
T. reesei, T. harzianum and A. niger fermentation were performed at different pH value (3.0, 4.0, 5.0, 6.0 7.0), temperature (20, 30 and 35 °C) and fermentation time (24, 48, 72 and 96 h). The following assay conditions were identical to those aforementioned.
Measurement of enzymes
The carboxymethyl cellulase (CMCase), filter paper activity (FPA) and β-glucosidase were measured as the description of Liao et al . All of experiments were performed in 0.05 M sodium acetate buﬀer (pH 4.8). 3, 5-dinitrosalicylic acid (DNS) reagent was used to measure the reducing sugar .
Pretreatment of ramie stalk
Air-dried ramie stalks from the Institute of Bast Fiber Crops at the Chinese Academy of Agricultural Sciences were cut into small chips (400–800 μm mesh). Three grams of ramie stalk was added into 7 mL H2O, 0.1% Tween 80, 4 mmol/L veratryl alcohol, 0.2 mmol/L Mn2+ in 300-mL Erlenmeyer flasks and sterilized at 121 °C for 20 min. P. eryngii were maintained on PDA plates at 4 °C and sub-cultured in PDA medium every month at 28 °C for 7 d. Four different P. eryngii pre-cultures were inoculated into the substrates, then incubated at 28 °C for 21 days. All of the experiments were carried out in triplicate. The content of cellulose, hemicellulose and lignin in pretreated substrate were 40%, 21% and 20%, respectively.
Enzymatic hydrolysis of pretreated ramie stalks were performed using Cellic ® CTec2 (Bagsvćrd, Denmark), the crude extracts of T. reesei, T. harzianum and A. niger (cultured at the optimal conditions) as enzymes (enzyme dosage was 30 FPU/g dry biomass). Enzyme cocktails were prepared as described in Table 1.
Enzymatic hydrolysis was carried out in 50 mL centrifuge tube for 48 h. Supernatant was collected after centrifuging sample at 10,000 rpm for 5 min to measure the reducing sugar.
All the experiments values presented in graphs and tables are mean ± SD, calculated using Excel 2017. Multiple comparison tests were performed with t test (significance levels = 0.05).
The conversion rate of reducing sugars (mg/g) were calculated as following:
Reducing sugars (mg/g) = reducing sugars obtained (mg) / biomass raw material (g)