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.

Lignocellulosic biomass, the most abundant renewable resource on earth, is considered as an alternative material to edible feedstocks for producing fermentable sugars, which can be further converted into value-added products including biofuels, bioplastics, and biochemicals. ^{1) 2) 3)} Lignocellulosic biomass mainly contains two structural polysaccharides, cellulose and xylan, as sources of fermentable sugars such as glucose and xylose. Generally, pretreatment steps that break down the rigid structure of the lignocellulosic matrix are required to enhance the accessibility of polysaccharide-hydrolyzing enzymes to the substrates. Pretreatment methods can be categorized as biological, chemical, physical, or physiochemical approaches. ^{4) 5) 6)} Alkali pretreatment is one of the chemical pretreatments that readily remove lignin and xylan side chains by the cleavage of ester bonds, resulting in improved efficiency of the subsequent enzymatic hydrolysis.

We developed a Ca(OH) ₂ -based alkali-pretreatment process called the “calcium capturing by carbonation (CO ₂ )” (CaCCO) process for sugar production from herbaceous feedstocks. ^{7) 8)} In this process, after the alkali pretreatment step, Ca(OH) ₂ is neutralized by carbonation to precipitate Ca ion as CaCO ₃ , which remains in the vessel during the subsequent enzymatic hydrolysis, and thus the solid–liquid separation step for washing the pretreated feedstock can be omitted. In a kg-scale conversion test of the CaCCO process, we adopted the enzymatic saccharification step under CO ₂ -pressurized conditions at a partial pressure of CO ₂ ( p CO ₂ ) of 0.9 MPa in order to lower the pH of the CaCO ₃ -containing slurry, resulting in the successful recovery of dense sugar solution. ⁹⁾ It is realized that the solubilization of CO ₂ by the pressurization would cause re-equilibration of the carbonate system by increasing the concentration of bicarbonate ion HCO ₃ ⁻ while decreasing that of carbonate ion CO ₃ ²⁻ . These changes in the distribution of species of dissolved inorganic carbon would result in an increase in proton concentration to decrease the pH. ¹⁰⁾ In the carbonate buffer system, CaCO ₃ would play an important role in keeping a constant pH by the reaction of the minerals with the excess acid. Meanwhile, the detailed effects of CO ₂ pressurization on the pH control and efficiency of enzymatic saccharification in the CaCCO process are not clear, due to heterogeneity of the reaction system and pH-dependent enzyme activities during saccharification. Herein, we investigated these effects using Ca(OH) ₂ -pretreated rice straw as a substrate and a mixture of fungal enzymes for saccharification.

Fine powder of sun-dried rice straw ( cv. Koshihikari), ¹¹⁾ whose cellulose and xylan contents of the absolutely dried rice straw powder were 35.0 and 14.1 %, respectively, was used as a feedstock. The experiment for investigating the effect of CO ₂ pressurization on the pH of slurry with Ca(OH) ₂ -pretreated/CO ₂ -neutralized (CaCCO-treated) rice straw powder was performed in a 2 L pressure-resistant reactor with a helical impeller (a custom-made reactor; Taiatsu Techno Corp., Tokyo, Japan). As shown in Fig. 1 , the pH of slurry decreased with increasing p CO ₂ . For example, pH values under the 0.1 and 1.0 MPa pressures of p CO ₂ were 6.38 and 5.70, respectively, in the case of the 5 % (w/w) slurry at 50 °C. Figure 1 also indicates that the pH values are affected by the temperature. The pH values at 40 °C are lower than those at 50 °C at the same p CO ₂ ; this can be explained by the higher solubility of CaCO ₃ at lower temperature. A similar trend was obtained in the 10 % (w/w) slurry, where pH values at 50 °C under the 0.1 and 1.0 MPa pressures of p CO ₂ were 6.51 and 5.64, respectively. All pH values of the slurry of pretreated rice straw powder were higher than those without biomass at the same p CO ₂ [“0 % 50 °C” in Fig. 1 ; the process was carried out using 10 % (w/w) Ca(OH) ₂ suspension]. Under the atmospheric conditions of p CO ₂ = 4 × 10 ⁻⁵ MPa, the pH of the slurry ranged between 8.3 and 8.6 (data not shown). Although we have not confirmed the pH at p CO ₂ lower than 0.1 MPa yet, we expect that the slurry pH of the CaCCO-treated feedstock could be controlled between 5.5 and 8.5 by changing the p CO ₂ from atmospheric levels to 1.0 MPa. In comparison to the case with concentrated acid or base solution added to the slurry to control the pH, CO ₂ pressurization with mechanical power would enable us to easily and reversibly control pH in both directions within the working range.

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.

Next, we carried out an enzymatic hydrolysis experiment of Ca(OH) ₂ -pretreated/water-washed rice straw powder to determine the pH range for effective saccharification. The substrate powder was prepared by HCl neutralization of the Ca(OH) ₂ -pretreated rice straw powder and washing of the solid part with water. ⁷⁾ Cellulose and xylan contents of this pretreated/washed rice straw powder were 35.8 and 15.6 %, respectively, on a dry basis. A mixture of two fungal enzyme preparations, namely, a cellulase preparation with high activity of hemicellulases from Trichoderma reesei M2-1 ¹²⁾ [12 filter-paper-degrading units (FPUs)/g-dry weight biomass at pH 5.0] and a commercial β-glucosidase preparation from Aspergillus niger [Novozyme 188, Novozymes Japan (Chiba, Japan); 43 cellobiase units (CbUs)/g-dry weight biomass at pH 5.0], was used as hydrolytic enzymes. The former preparation was obtained by the cultivation of M2-1 with continuous feeding of a mixed solution of glucose, xylose, and cellobiose described by Ike et al . ¹³⁾ Figure 2 shows the sugar yields after enzymatic saccharification under various pH conditions. More soluble sugars from glucan and xylan were yielded at conditions of pH 5.0–6.0 and pH around 5.5–6.0, respectively. At pH above 6.0, the sugar yields decreased and the liberated monosaccharides in particular were at a significantly low level at pH 7.0. These results indicate that pH of 5.5–6.0 is suitable for the saccharification of Ca(OH) ₂ -pretreated/water-washed rice straw powder.

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. ¹³⁾

Next, we examined the enzymatic saccharification of CaCCO-treated rice straw powder under the CO ₂ -pressurized conditions to investigate the effect of p CO ₂ on the sugar yield after enzymatic saccharification. As shown in Fig. 3 , the saccharification ratio increased as the p CO ₂ increased. The maximum amount of total liberated sugars from glucan and xylan (mono- and oligosaccharides) was achieved at the p CO ₂ of 0.4 MPa and over, while that of monosaccharides (glucose and xylose) was obtained at the p CO ₂ of 0.5 MPa. Enzymatic saccharification in this experiment was performed at the slurry concentration of 5 % (w/w) and 50 °C; the pH of the slurry at the p CO ₂ of 0.4–0.5 MPa under these saccharification conditions is expected to be 6.0, according to the data in Fig. 1 . This value is consistent with the result of Fig. 2 that pH of 5.5–6.0 is suitable for enzymatic saccharification.

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.

In this study, a mixture of enzymes from two fungi was adopted for saccharification; the optimal pH for its cellulolytic (filter-paper-degrading and endoglucanase) and xylanolytic (birchwood-xylan-degrading) activities was 5.0 (data not shown). Meanwhile, T. reesei possesses at least six genes for xylanases, and two xylanases, XYN II and XYN III, which are major xylanases in the cellulase preparation, possess the optimum pH of 5.5–6.0. ^{14) 15) 16) 17) 18)} It has also been reported that xylan removal strongly affects the efficiency of cellulose hydrolysis, and there is synergy between some types of cellulases and xylanases for the degradation of lignocellulosic biomass. ^{19) 20) 21) 22) 23)} It is speculated that some enzymes such as XYN II and XYN III might contribute more significantly to hydrolysis at pH 5.5–6.0 than at other pH levels, which could increase the accessibility of cellulases to the substrate, resulting in the maximum sugar yield.

In summary, we elucidated the effect of CO ₂ pressurization on pH control and enzymatic saccharification of Ca(OH) ₂ -pretreated rice straw powders, and determined the minimum pressure for saccharification with fungal enzymes. Taking into account that this CO ₂ -pressurizing system could control pH of the slurry between 5.5 and 8.5, potential applications of this system would include the use of other enzymes with pH optima within the controllable range, the use of two-step conversion of substrate with two kinds of enzymes with distinct pH optima (like α-amylase and glucoamylase for starch saccharification), simple pH-controlled bioconversion and/or slurry circulation without oxygen, the use of CO ₂ in solution as substrate for its fixation, and dynamic pH shifts for changing the solubility of contaminants or products like lignin and polyphenols. While the effect of solubilization of calcium ions in the presence of large amounts of CO ₂ in solution was not obvious in this study, the potential impact of ionic strength and/or direct effects of calcium ions on bio-catalytic activities should be considered for maximizing the yields.

CONFLICTS OF INTEREST

The authors declare no conflict of interests.