Synergistic Lignocellulose Hydrolysis

For hydrolysis by the enzyme of C. owensensis alone, each reaction system was prepared in 50 mM sodium acetate, pH 6.0 with the dry substrate of 2 % (w/v) and the enzyme loading of 15 mg protein per gram dry substrate. The reaction volume was 500 μl in a 2 ml Eppendorf tube, which was sealed by winding parafilm after closing the lip, and put in a water bath at 70 °C for 48 h.
For synergetic hydrolysis by the enzyme of C. owensensis and the commercial enzyme cocktail Cellic CTec2 (Novoyzmes), two trials were performed. One was that the lignocellulosic biomass (native corn stover or native corncob) was sequentially hydrolyzed (SH) by the enzyme of C. owensensis (the first step) and CTec2 (the second step). The first step was the same as described above (for hydrolysis by the enzyme of C. owensensis only). After 48 h hydrolysis by the enzyme of C. owensensis (the first step) the CTec2 and 500 μl of sodium acetate buffer, pH 5.0 were added, forming a reaction system of pH 5.0 with the dry substrate of 1 %, and then incubated in water bath at 50 °C for 72 h (the second step). The other was that the lignocellulosic biomass was co-hydrolyzed (CH) by the enzyme of C. owensensis and CTec2 in the sodium acetate buffer of pH 5.0 with the dry substrate of 1 % at 50 °C for 72 h. The loading rates of CTec2 (http://www.bioenergy.novozymes.com/) for synergetic hydrolysis were 30 mg/g glucan (high loading). These lignocellulosic biomasses were hydrolyzed by CTec2 alone in the sodium acetate buffer of pH 5.0 with the dry substrate of 1 % at 50 °C for 72 h as controls.
Reducing sugar assay was carried out by PHBAH method with xylose as the standard [51 (link), 52 (link)]. Glucose and xylose concentrations were measured on a HPLC system equipped with a Hi-Plex Ca column (7.7 × 300 mm, Agilent Technology, USA), LC-20AT pump (Shimadzu, Japan) and RID-10A refractive index detector (Shimadzu, Japan), using water at a flow rate of 0.6 ml/min as mobile phase. The amounts of released glucose and xylose were used for calculating glucan and xylan conversions, respectively. The furfural and 5-hydroxymethyl furfural (HMF) were analyzed by HPLC as described above. The phenolics in the hydrolysate were analyzed by ultraviolet spectra at 280 nm using p-hydroxy benzaldehyde as the standard.
The native corn stover morphologies before and after hydrolysis and after incubated in the acetate buffer (pH 6.0) at 70 °C for 48 h were examined by scanning electron microscopy (SEM). The specimens were mounted on stubs and sputter-coated with gold prior to imaging with a JEOL JSM-6700F scanning electron microscope using 5-kV accelerating voltage and 10-mm distance.