Control of pH by CO 2 Pressurization for Enzymatic Saccharification of Ca(OH) 2 -Pretreated Rice Straw in the Presence of CaCO 3

Abstract

The aim of this study was to investigate the effect of pH control by CO ₂ pressurization on the enzymatic hydrolysis of herbaceous feedstock in the calcium capturing by carbonation (CaCCO) process for fermentable sugar production. The pH of the slurry of 5 % (w/w) Ca(OH) ₂ -pretreated/CO ₂ -neutralized rice straw could be controlled between 5.70 and 6.38 at 50 °C by changing the CO ₂ partial pressure ( p CO ₂ ) from 0.1 to 1.0 MPa. A mixture of fungal enzyme preparations, namely, Trichoderma reesei cellulases/hemicellulases and Aspergillus niger β-glucosidase, indicated that pH 5.5-6.0 is optimal for solubilizing sugars from Ca(OH) ₂ -pretreated rice straw. Enzymatic saccharification of pretreated rice straw under various p CO ₂ conditions revealed that the highest soluble sugar yields were obtained at p CO ₂ 0.4 MPa and over, which is consistent with the expected pH at the p CO ₂ without enzymes and demonstrates the effectiveness of pH control by CO ₂ pressurization.

Keywords: CO 2 -pressurized enzymatic saccharification; calcium capturing by carbonation (CaCCO) process; fungal cellulase; fungal β-glucosidase.

Fig. 1.. Effect of CO ₂ partial pressure ( p CO ₂ ) on pH of slurry with the CaCCO-treated rice straw powder.

A mixture of rice straw powder [ cv . Koshihikari, 0.5-mm-mesh pass; 0 g for 0 % (w/w), 90 g for 5 % (w/w), 180 g for 10 % (w/w)], Ca(OH) ₂ (18 g), and distilled water [1.8 L for 0 % (w/w), 1.71 L for 5 % (w/w), 1.62 L for 10 % (w/w)] was heated at 120 °C for 90 min. After cooling, the slurry was poured into a 2 L pressure-resistant reactor with a helical impeller. The reactor was then immersed in a heated water bath to keep the temperature of the slurry at an appropriate level (40 or 50 °C), and the CO ₂ was injected into the reactor for neutralization of the slurry until its pH was equilibrated. The pH of the slurry was monitored using the sensor InPro4800SG/225/PT1000 (Mettler Toledo, Tokyo, Japan), which was installed with the reactor. After neutralization, the reactor was pressurized up to the desired partial pressure of CO ₂ and the pH was measured after equilibrium was achieved.

Fig. 2.. Sugar yields after 24 h of enzymatic saccharification of Ca(OH) ₂ -pretreated/water-washed rice straw powder under various pH conditions.

The Ca(OH) ₂ -pretreated/water-washed pretreated rice straw (50 mg on a dry basis) was added to 1 mL of enzyme solution in the 50 mM buffer with different pH (acetate buffer pH 4.0, 4.5, 5.0, 5.5, and 6.0; phosphate buffer pH 6.0, 6.5, and 7.0). A mixture of the cellulase preparation from T. reesei M2-1 (0.6 FPU at pH 5.0) and Novozyme188 (2.2 CbU at pH 5.0) was used as saccharification enzyme. All saccharification reactions were performed at 50 °C for 24 h. The amounts of liberated monosaccharides (glucose and xylose) and solubilized sugars (glucose + glucose-containing oligosaccharides and xylose + xylose-containing oligosaccharides) during saccharification were measured by HPLC, as described in our previous report.

Fig. 3.. Sugar yields after 24 h of CO ₂ -pressurized enzymatic saccharification of CaCCO-treated rice straw powder.

All reactions including Ca(OH) ₂ pretreatment, CO ₂ neutralization, and enzymatic saccharification were carried out in the 96 mL pressure-resistant glass tube "Hiper Glass Cylinder (HGC)" (HPG-96-3; Taiatsu Techno Corp.). One gram of fine-powdered rice straw, 100 mg of Ca(OH) ₂ , and 18 mL of water in the HGC were well mixed and heated at 120 °C for 1 h. After cooling, CO ₂ was injected into the HGC at a pressure of 0.5 MPa and settled at room temperature overnight to complete neutralization of the slurry. Then, 1 mL of the same enzyme mixture in Fig. 2 was added to the neutralized slurry in the HGC, followed by pressurization of CO ₂ up to the desired partial pressure. Saccharification reactions were performed at 50 °C for 24 h and the amounts of liberated monosaccharides and solubilized sugars were measured in the same way as for Fig. 2.