Energy Fuels 2011, 25, 438–445 Published on Web 12/03/2010
: DOI:10.1021/ef101218a
Biomimetic Sequestration of CO2 and Reformation to CaCO3 Using Bovine Carbonic Anhydrase Immobilized on SBA-15 Mari Vinoba,†,^ Dae Hoon Kim,†,‡ Kyoung Soo Lim,† Soon Kwan Jeong,*,† Seung Woo Lee,§ and Muthukaruppan Alagar ^ † Korea Institute of Energy Research, Daejeon 305-343, Korea, ‡Department of Chemical and Biological Engineering, Korea University, Seoul 136-713, Korea, §Korea Institute of Geoscience and Mineral Resources, Daejeon 305-350, Korea, and ^ Department of Chemical Engineering, Anna University Chennai, Chennai 600- 025, India
Received September 8, 2010. Revised Manuscript Received November 13, 2010
The biocatalytic capture of CO2, and its precipitation as CaCO3, over bovine carbonic anhydrase (BCA) immobilized on a pore-expanded SBA-15 support was investigated. SBA-15 was synthesized using TMB as a pore expander, and the resulting porous silica was characterized by XRD, BET, IR, and FE-SEM analysis. BCA was immobilized on SBA-15 through various approaches, including covalent attachment (BCA-CA), adsorption (BCA-ADS), and cross-linked enzyme aggregation (BCA-CLEA). The effects of pH, temperature, storage stability, and reusability on the biocatalytic performance of BCA were characterized by examining para-nitrophenyl acetate (p-NPA) hydrolysis. The Kcat/Km values for p-NPA hydrolysis were 740.05, 660.62, and 680.11 M-1 s-1, respectively, whereas the Kcat/Km value for free BCA was 873.76 M-1 s-1. The amount of CaCO3 precipitate was measured quantitatively using an ion-selective electrode and was found to be 12.41, 11.82, or 11.28 mg CaCO3/mg for BCA-CLEA, BCA-ADS, or BCACA, respectively. The present results indicate that the immobilized BCA-CLEA, BCA-ADS, and BCA-CA are green materials, and are tunable, reusable, and promising biocatalysts for CO2 sequestration.
organo-silicas, surface-modified silicas,6-10 and amine or alkanolamines.11,12 However, development of commercial CO2 capture technologies is expensive, energy-intensive, and results in equipment corrosion, thereby reducing plant efficiency.13 Currently, novel biomimetic approaches to CO2 sequestration based on the enzyme bovine carbonic anhydrase (BCA) as a biocatalyst have attracted attention, and a proof of principle has already been demonstrated.14,15 The biocatalytic sequestration system offers several potential advantages: plant-byplant solutions for emissions reduction; the method does not require costly CO2 concentration and transportation steps; the method is safe, stable, and yields environmentally benign products; and the process is environmentally benign.14 For instance, CO2 sequestration to CaCO3 using various industrial brines containing Ca2þ or Mg2þ cations over carbonic anhydrase (CA) immobilized in chitosan-alginate beads has been reported.16 Similarly, Mirjafari et al. studied the precipitation of CaCO3 from a model brine made by aqueous dissolution of CaCl2 3 2H2O in a buffer solution containing the free CA enzyme.17 A previous report stated that the biocatalytic
1. Introduction The most significant means of energy generation throughout the world is the combustion of fossil fuels, which results in emission of the greenhouse gas CO2. The accumulation of greenhouse gases in the atmosphere leads to global warming; hence, huge efforts are targeted at mitigating the accumulation of such gases. Recent growth of economy and increasing population have contributed to the ever-increasing atmospheric CO2 levels. Hence, the necessity to control CO2 concentrations in the atmosphere without reducing the use of fossil fuels presents a challenge for researchers. A large variety of CO2 sorbents have been reported in the literature, including oxides,1 zeolites,2 activated carbon,3 metal-organic frameworks,4,5 *To whom correspondence should be addressed. E-mail: jeongsk@ kier.re.kr. Mail: Clean Fossil Research Center, Climate Change Technology Research Division, Korea Institute of Energy Research, 102 Gajeong-ro, Yuseong-gu, Daejeon 305-343, Korea. (1) Lee, K. B.; Beaver, M. G.; Caram, H. S.; Sircar, S. Ind. Eng. Chem. Res. 2008, 47, 8048–8062. (2) Cavenati, S.; Grande, C. A.; Rodrigues, A. E. J. Chem. Eng. Data 2004, 49, 1095–1101. (3) Himeno, S.; Komatsu, T.; Fujita, S. J. Chem. Eng. Data 2005, 50, 369–376. (4) Millward, A. R.; Yaghi, O. M. J. Am. Chem. Soc. 2005, 127, 17998–17999. (5) Llewellyn, P. L.; Bourrelly, S.; Serre, C.; Filinchuk, Y. T.; Ferey, G. Angew. Chem. Int. Ed. 2006, 45, 7751–7754. (6) Harlick, P. J. E.; Sayari, A. Ind. Eng. Chem. Res. 2007, 46, 446– 458. (7) Hicks, J. C.; Drese, J. D.; Fauth, D. J.; Gray, M. L.; Qi, G.; Jones, C. W. J. Am. Chem. Soc. 2008, 130, 2902–2903. (8) Huang, H. Y.; Yang, R. T.; Chinn, D.; Munson, C. L. Ind. Eng. Chem. Res. 2003, 42, 2427–2433. (9) Xu, X.; Song, C.; Miller, B. G.; Scaroni, A. W. Ind. Eng. Chem. Res. 2005, 44, 8113–8119. (10) Chaffee, A. L.; Knowles, G. P.; Liang, Z.; Zhang, J.; Xiao, P.; Webley, P. A. Int. J. Greenhouse Gas Control 2007, 1, 11–18. r 2010 American Chemical Society
(11) Puxty, G.; Rowland, R.; Allport, A.; Yang, Q.; Bown, M.; Burns, R.; Maeder, M.; Attalla, M. Environ. Sci. Technol. 2009, 43, 6427–6433. (12) Guerrero, R. S.; Dana, E.; Sayari, A. Ind. Eng. Chem. Res. 2008, 47, 9406–9412. (13) Veawab, A.; Tontiwachwuthikul, P.; Chakma, A. Ind. Eng. Chem. Res. 1999, 38, 3917–3924. (14) Bond, G. M.; Egeland, G.; Brandvold, D. K.; Medina, M. G.; Simsek, F. A.; Stringer, J. World Resour. Rev. 1999, 11, 603–619. (15) Bond, G. M.; Stringer, J.; Brandvold, D. K.; Simsek, F. A.; Medina, M. G.; Egeland, G. Energy Fuels 2001, 15, 309–316. (16) Liu, N.; Bond, G. M.; Abel, A.; McPherson, B. J.; Stringer, J. Fuel Process. Technol. 2005, 86, 1615–1625. (17) Mirjafari, P.; Asghari, K.; Mahinpey, N. Ind. Eng. Chem. Res. 2007, 46, 921–926.
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Energy Fuels 2011, 25, 438–445
: DOI:10.1021/ef101218a
Vinoba et al.
capture of CO2 using CA and its transformation to solid carbonate operated optimally under basic conditions.18 The enzyme activity in basic solutions was found to be more efficient than in acidic conditions. Based on the above reports, we studied the precipitation of CaCO3 produced by BCA immobilized on mesoporous silica under basic conditions. Here, the hydration of CO2 and sequestration as CaCO3 were carried out at pH 8 and 10.0, respectively.19 The major drawbacks to the use of immobilized enzyme were overcome by performing the hydration of CO2 in a fixed bed followed by precipitation in the collector. Thus, the tedious steps of catalyst separation and cleaning of the spent catalyst were avoided, which allowed for continuous hydration and sequestration of CO2 to CaCO3. The present study demonstrates the immobilization of the enzyme BCA onto mesoporous silica through various techniques, and the catalytic activity of BCA was tested by examining the hydrolysis of para-nitrophenyl acetate (p-NPA). This study was extended to test the biocatalytic activity of immobilized BCA measured by the quantity of precipitated CaCO3 after hydration of CO2.
an aqueous buffer (100 mM sodium phosphate buffer, pH 7.0) then incubated in a sodium phosphate buffer containing 0.1% GA at 150 rpm with shaking. After GA treatment for 30 min, the samples were washed with copious amounts of Tris buffer (50 mM pH 8.0) then stored at 4 C. The final product was designated BCA-CLEA. BCA immobilization by the ADS method was performed by a procedure that was similar to that described for preparing CLEA-BCA without the GA treatment step, and the product was denoted BCA-ADS. In the case of BCA immobilization by the CA method, aldehyde-functionalized SBA-15 was prepared by incubating SBA-15/APTES in a 0.1% GA solution in 50 mM sodium phosphate, (pH 8.0) for 1 h. Subsequently, the aboverecovered product was treated with BCA by a procedure similar to that of the CLEA method to obtain BCA-CA. The amount of attached and leached BCA was calculated by the Bradford method.23 2.4. Hydrolysis of p-NPA using BCA-CLEA, BCA-ADS, and BCA-CA. The enzyme activities of BCA-CLEA, BCA-ADS, and BCA-CA were estimated spectrophotometrically using p-NPA as a substrate, as reported, with slight modification.24,25 Here, enzyme activities were measured at 25 C by monitoring the changes in concentration of p-NP, which is one of the hydrolysis products of p-NPA. For the free BCA, the activity assay was performed in a 1-mL UV cuvette. The reaction mixture, composed of 0.8 mL of Tris Buffer (50 mM, pH 8), 0.1 mL of substrate solution (p-NPA dissolved in acetonitrile), and 0.1 mL of enzyme solution, was mixed in the cuvette using a micropipet. The final mixture contained 10% acetonitrile. The enzyme activity was measured in an Optizen 2120F UV/vis spectrophotometer at 400 nm. The free enzyme activity was determined by varying the concentration (0.5, 1, 1.5, 2.0, and 2.5 mM) of the substrate while holding the enzyme concentration constant, or by varying the enzyme concentration (0.2, 0.4, 0.6, 0.8, and 1.0 μM) while holding the substrate concentration constant. Blank experiments were also conducted to estimate the self-dissociation of p-NPA in each assay solution. Similarly, immobilized enzyme activity was measured by mixing 18 mL of Tris- HCl buffer (50 mM, pH 8), 2 mL of substrate solution, and an appropriate amount of BCA-CLEA, BCA-ADS, and BCA-CA in a beaker. At intervals of 1 min over a period of 15 min, 1-mL aliquots of the solution were drawn from the reaction mixture and the absorbance was measured at 400 nm. The immobilized enzyme activity was determined by varying the concentration (0.5, 1, 1.5, 2.0, and 2.5 mM) of the substrate while holding the mass of the catalyst constant, or by varying the mass of the catalyst (1-5 mg) while holding the substrate concentration constant. The same catalyst was washed with Tris-HCl buffer and used for later studies. 2.5. CO2 Sequestration and Evaluation of CaCO3. CaCO3 precipitation was carried out after hydration of CO2 in a fixed bed formed in a glass tube. Typically, 25 mg of immobilized enzyme, containing 5.226, 3.70, and 2.175 mg of BCA, in BCACLEA, BCA-ADS, and BCA-CA, respectively, were packed in a glass tube equipped with an infusion pump, and the catalytic bed temperature was maintained at 30 C. A saturated CO2 solution was introduced at a flow rate of 0.9 mL/min and was mixed with 0.1 mL/min 0.5 M Tris-HCl buffer (pH 8.0) for 150 min before reaching the catalytic bed. The hydrated CO2 (HCO3-) was collected in a beaker containing 40 mL of 4% CaCl2 solution in 0.5 M Tris base (pH 10.0) and was subsequently precipitated as CaCO3. Furthermore, hydration of CO2 was performed using a free enzyme solution (5 mg) at pH 8, and the product was precipitated as CaCO3 using 40 mL of 4% CaCl2 solution in 0.5 M Tris base (pH 10.0). The carbonate obtained was filtered, dried
2. Experimental Section 2.1. Materials. Bovine carbonic anhydrase (BCA), tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxy silane (APTES), P123 (triblock copolymer (poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide), EO20-PO70-EO20, MW 5800)), hydrochloric acid (HCl, 37%), trimethyl benzene (TMB), glutaraldehyde (GA), 2-amino 2-(hydroxymethyl)-1,3propanediol (Tris base), p-nitrophenol (p-NP), p-nitrophenyl acetate (p-NPA), acetonitrile, anhydrous calcium chloride, and protein assay reagents for producing the Bradford reagent were purchased from Aldrich and used without purification. All solutions were prepared with deionized water. 2.2. Synthesis of Amine-Functionalized Mesoporous SBA-15. Mesoporous silica SBA-15 was synthesized as per the reported procedure.20 The molar composition of the reaction mixture was 1:0.017:5.87:0.0025:183 TEOS/P123/HCl/TMB/H2O. Briefly, 4 g of pluronic P123 was dissolved in 150 mL of 1.6 M HCl at 40 C, 0.3 g of TMB and 9.2 mL of TEOS were then added. The mixture was stirred at 40 C for 8 h then aged at 80 C for 24 h. The recovered as-synthesized sample was calcined in air at 550 C for 6 h with a heating rate of 1 C/min. Amine-functionalized SBA-15 was obtained by refluxing 1 g of SBA-15 and 5 mM APTES solution in dry toluene (100 mL) under a N2 atmosphere for 24 h. The product was then filtered, washed with ethanol, and dried overnight at 80 C under vacuum. The obtained material was designated SBA-15/APTES. 2.3. Enzyme Immobilization over the Amine-Functionalized SBA-15. BCA immobilization over the mesoporous silica was performed by three methods via cross-linked enzyme aggregation (CLEA), adsorption (ADS), and covalent attachment (CA).21,22 Typically, CLEA of BCA was achieved by mixing 10 mg of SBA-15 with 2 mL of free BCA in buffer (3 mg/mL BCA in 100 mM sodium phosphate, pH 7.0) and incubating at 25 C with shaking. After 1 h incubation to allow for BCA adsorption, the samples were washed with minimum amounts of (18) Favre, N.; Christ, M. L.; Pierre, A. C. J. Mol. Catal. B., Enzym. 2009, 60, 163–170. (19) Appelo, C. A. J.; Postman, D. Geochemistry, Groundwater and Pollution, 2nd ed.; Taylor & Francis: New York, 2005; pp 176-178. (20) Zhao, D.; Feng, J.; Huo, Q.; Melosh, N.; Fredrickson, G. H.; Chmelka, B. F.; Stucky, G. D. Science 1998, 279, 548–552. (21) Kim, M. I.; Kim, J.; Lee, J.; Jia, H.; Na, H. B.; Youn, J. K.; Kwak, J. H.; Dohnalkova, A.; Grate, J. W.; Wang, P.; Hyeon, T.; Park, H. G.; Chang, H. N. Biotechnol. Bioeng. 2007, 96, 210–218. (22) Kim, M. I.; Kim, J.; Lee, J.; Shin, S.; Na, H. B.; Hyeon, T.; Park, H. G.; Chang, H. N. Microporous Mesoporous Mater. 2008, 111, 18–23.
(23) Bradford., M. M. Anal. Biochem. 1976, 72, 248–254. (24) Crumbliss, A. L.; McLachlan, K. L.; Odaly, J. P.; Henkens, R. W. Biotechnol. Bioeng. 1988, 31, 796–801. (25) Ozdemir, E. Energy Fuels 2009, 23, 5725–5730. (26) Al-Khaldi, M. H.; Nasr-El-Din, H. A.; Mehta, S.; Al-Aamri, A. D. Chem. Eng. Sci. 2007, 62, 5880–5896.
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: DOI:10.1021/ef101218a
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Table 1. Textural Properties of SBA-15 Before and After Immobilizationa sample
SBET (m2/g)
Vp (cm3/g)
Dp (nm)
SBA-15 SBA-15/APTES BCA-ADS BCA-CA BCA-CLEA
633 518 286 327 213
1.42 1.32 0.76 0.67 0.63
15.2 11.2 6.2 7.2 5.6
a
SBET, surface area; Vp, mean pore volume; Dp, pore diameter.
at 60 C, and submitted to characterization. The quantity of carbonate precipitate was estimated using a carbon dioxide ion selective electrode (CISE) (Thermo Scientific, Orion 9502BNWP). This electrode was calibrated with a standard CaCO3 solution. Here, 15 mg of the precipitated CaCO3 was treated with 10 mL of CO2 buffer at pH 5.0 (a mixture of Tris sodium citrate, citric acid, sodium chloride, deionized water, cat. no. Orion 950210) and the volume was brought to 100 mL by addition of DI water. The liberated CO2 was quantified by CISE. The amount of liberated CO2 was equivalent to the amount of CaCO3 precipitated. The reaction between the CO2 buffer and CaCO3 was as follows:26 Citric Acid þ Calcium Carbonate þ Water
Figure 1. FT-IR spectra of SBA-15 and SBA-15/APTES.
(isotherms with hysteresis). The nitrogen adsorption-desorption isotherms for all (before and after APTES grafting) mesoporous SBA-15 were type-IV isotherms. SBA-15 and SBA15/APTES (functionalized SBA-15) exhibited a well-defined hysteresis loop between the partial pressures of P/P0=0.68-0.75, indicating a mesophase. The BET results suggested that SBA-15/APTES showed a decrease in the specific area corresponding to the pore volume and pore diameter relative to SBA-15. Such a significant decrease in the textural properties of SBA-15/APTES was due to pore filling and/or structural construction of APTES, which was in line with the XRD results.27 The FT-IR spectra of SBA-15 and SBA-15/APTES are shown in Figure 1. The disappearance of the band at 966 cm-1 (representing the bending modes of SiOH) indicated that the silanol groups were reduced upon APTES grafting. The corresponding appearances of new bands at 2935 and 2816 cm-1 were attributed to the asymmetric and symmetric stretching modes of the -CH2 APTES groups. Bands were identified at 1560 cm-1 (δN-H), -NH2 (νas =3438 cm-1, νs = 3427 cm-1), and 694 cm-1 (δ C-H). These results confirmed the successful functionalization of SBA-15 with APTES.28 Figures 2A and 2B depict the 13C MAS NMR and 29Si MAS NMR spectra of the parent SBA-15 and SBA-15/APTES, respectively. 13C MAS NMR spectra of SBA-15/APTES (Figure 2A) showed three signals at 43.51, 23.67, and 9.96 ppm, attributed to the presence of -CH2-NH2, -CH2-, and -CH2-Si, respectively, which provided evidence for the grafting of APTES onto SBA-15. 29 The 29 Si MAS NMR spectra (Figure 2B) showed two peaks at -101.3 and -109.3 ppm that were commonly observed in all mesoporous SBA-15 materials and were attributed to Q3(Si(OSi)3(OH)) and Q4(Si(OSi)4) silicon atoms, respectively. New peaks emerged at -59 and 67 ppm, which were assigned to the T2(Si(OSi)2R(OH)1) and T3(Si(OSi)4) silicon species formed during the reaction between defective Si-OH groups of mesoporous silica and APTES.30
¼ Calcium Citrate þ Carbon dioxide þ Water 2.6. Characterization. Powder X-ray diffraction patterns (XRD) were recorded using a Rigaku Miniflex diffractometer with Cu KR radiation (λ=0.154 nm). The diffraction data were recorded in the 2θ range 0.5-10 at a 0.02 step size with 1 s step time. The nitrogen adsorption-desorption isotherms were measured at -196 C on a Micromeritics ASAP 2010 volumetric adsorption analyzer. Prior to each adsorption measurement, the samples were evacuated at 100 C under vacuum (p
