2.4 Pretreatment of pine wood
Pine wood (Pinus densiflora , diameter: 13 cm) was chopped into approximately 0.25 cm (width) × 0.35 cm (height) × 4.5 cm (length) chips, and 100 g L-1 of the wood chips were soaked in hydrogen peroxide (H2O2): acetic acid (CH3COOH) solution (1:1 ratio, HPAC solution).[33] The wood chip sample was delignified in a water bath at 80 °C for 2–3 h. The delignified wood chips were then strained and thoroughly washed with water until the HPAC solution was completely removed. Finally, the sample was freeze dried and stored at room temperature.
2.5 Enzyme preparation and hydrolysis
Cellulase was produced using the Rut-C30 strain of Trichoderma reesei .[22] The activity of the cellulase stock on filter paper was measured to be 50 FPU mL-1. Xylanases derived from Thermomyces lanuginosus (Cat.X2753-50G, St. Louis, MI, USA) was purchased from Sigma-Aldrich. β-glucosidase ofAspergillus niger (Lot 141001, Wicklow, Ireland) was obtained from Megazyme. One unit of xylanase was defined as the enzyme concentration that released 5 g L-1 of reducing sugars from 1% beechwood xylan (X4252-100G, Sigma-Aldrich) at 50 °C for 10 min. β-glucosidase, 20 µg mL-1, was completely hydrolyzed to glucose in 6.85 g L-1 cellobiose over 10 min at 50 °C, this amount was defined as one unit.
The HPAC-pretreated pine was weighed to prepare 1–5, and 10% (g v-1) in 100 mL citric acid buffer (10 mM, pH 5.5) and hydrolyzed with 20 FPU cellulase g-1 biomass and auxiliary enzymes (200 units L-1 xylanase and 100 units L-1 β-glucosidase). In the case of the small scale volume, 2% HPAC-pretreated pine was prepared in 1 mL citrate buffer (10 mM, pH 5.5) with 5−100 FPU g-1 biomass alongside 2 units of xylanase and 1 unit of β-glucosidase. All the reactions were performed at 50 °C for 12–96 h. The solutions were centrifuged at 13000 rpm for 10 min to obtain a clear hydrolysate. The concentrations of fermentable sugars were measured using a DNS assay and HPLC analysis. [21, 33] To prepare the feedstock for fed-batch system, 20% HPAC-pretreated pine was hydrolyzed with 20 FPU cellulase g-1 biomass along with the auxiliary enzymes at 50 °C for one week. The supernatant was clarified through filtration and then incubated more for 24 h. The concentration of reducing sugar in the hydrolysate was measured as 155.08 g L-1.
All of the hydrolysates were stored at -20 °C until used for succinic acid fermentation.