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.