Article pubs.acs.org/est
One-Pot Enzymatic Conversion of Carbon Dioxide and Utilization for Improved Microbial Growth Sung-Gil Hong,†,# Hancheol Jeon,‡,# Han Sol Kim,† Seung-Hyun Jun,† EonSeon Jin,*,‡ and Jungbae Kim*,†,§ †
Department of Chemical and Biological Engineering, Korea University, Seoul 136-701, Republic of Korea Department of Life Science, Research Institute for Natural Sciences, Hanyang University, Seoul, 133-791, Republic of Korea § Green School, Korea University, Seoul 136-701, Republic of Korea ‡
S Supporting Information *
ABSTRACT: We developed a process for one-pot CO2 conversion and utilization based on simple conversion of CO2 to bicarbonate at ambient temperature with no energy input, by using the cross-linking-based composites of carboxylated polyaniline nanofibers (cPANFs) and carbonic anhydrase. Carbonic anhydrase was immobilized on cPANFs via the approach of magnetically separable enzyme precipitate coatings (Mag-EPC), which consists of covalent enzyme attachment, enzyme precipitation, and crosslinking with amine-functionalized magnetic nanoparticles. Mag-EPC showed a half-life of 236 days under shaking, even resistance to 70% ethanol sterilization, and recyclability via facile magnetic separation. For one-pot CO2 conversion and utilization, Mag-EPC was used to accelerate the growth of microalga by supplying bicarbonate from CO2, representing 1.8-fold increase of cell concentration when compared to the control sample. After two repeated uses via simple magnetic separation, the cell concentration with Mag-EPC was maintained as high as the first cycle. This one-pot CO2 conversion and utilization is an alternative as well as complementary process to adsorption-based CO2 capture and storage as an environmentally friendly approach, demanding no energy input based on the effective action of the stabilized enzyme system.
1. INTRODUCTION
recycled uses of enzymes to improve the economics of enzymatic conversion and follow-up utilization of CO2. During the last decade, we have witnessed a series of successes in stabilizing enzyme activity by using nanostructured materials for their immobilization. Nanoparticles, nanofibers, nanotubes, and nanoporous materials provide a large surface area or high pore volume to achieve high enzyme loadings, and the approach of chemical enzyme cross-linking has demonstrated its great potential in stabilizing the enzyme activity in an unprecedented ways.14 For example, an enzyme called trypsin was immobilized on electrospun polymer nanofibers in the form of cross-linked enzyme coatings, achieving 200-fold higher enzyme loading than the conventional approach of covalent enzyme attachment and showing negligible activity decrease at shaking condition under repetitive uses for one year.15 In the present work, we first report one-pot conversion and utilization of CO2 based on a highly stabilized and magnetically separable enzyme system, which is even resistant to the ethanol sterilization. Carbonic anhydrase and magnetic nanoparticles
Outpacing CO2 emission in the carbon cycle has fueled a great interest in CO2 capture and storage (CCS) as a potential solution for atmospheric CO2 reduction.1,2 The approach of CO2 adsorption using various adsorbent solvents has been proposed and used for the process of CO2 capture. However, high energy input for CO2 desorption from adsorbents is a major drawback of adsorption-based CO2 capture.3−5 Even though various alternative approaches using metal−organic frameworks (MOFs), activated carbons, and mesoporous materials have been proposed,6,7 an economical burden of high energy input for CO2 desorption is still a critical issue against the practical implementation of CCS. As a potential solution, an enzyme, called carbonic anhydrase, can directly convert CO2 to bicarbonate (HCO3−) via its catalytic reaction at a high turnover rate such as 106 per second.8 This enzymatic CO2 conversion does not require high energy input at all, and the bicarbonate can be further utilized not only for the production of carbonate salts9,10 and methanol,11 but also for the growth of photosynthetic organisms12,13 that can produce value-added chemicals and biodiesels. However, the success of enzymatic CO2 conversion requires the successful immobilization and stabilization of carbonic anhydrase, which enable the © XXXX American Chemical Society
Received: October 21, 2014 Revised: March 19, 2015 Accepted: March 19, 2015
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Figure 1. Schematic illustrations for one-pot conversion and utilization of CO2 by accelerated growth of photosynthetic organisms.
2.3. Immobilization of Carbonic Anhydrase on Carboxylated Polyaniline Nanofibers. Carbonic anhydrase was immobilized on cPANFs by four different approaches: covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetically separable EPC (Mag-EPC) (Figure 2). Carbonic anhydrase solution (10 mg/ mL) in 100 mM sodium phosphate buffer (PB, pH 7.6) was centrifuged at 10 000 rpm for 10 min, and filtered by using a syringe filter (0.22 μm pore size) to eliminate insoluble impurities. cPANF prepared at the molar ratio of 0.25:0.75 (aniline: 3-ABA) was used for the immobilization of carbonic
were coimmobilized via an approach of magnetically separable enzyme precipitate coatings (Mag-EPC) on carboxylated polyaniline nanofibers (cPANFs). After sterilization in 70% ethanol solution, Mag-EPC was added to the culture of photosynthetic organisms, which are biocatalysts for the generation of biodiesels and value-added chemicals (Figure 1). Mag-EPC can effectively convert atmospheric CO2 to bicarbonate, expediting the growth of photosynthetic organisms, and be recycled via facile magnetic separation.
2. MATERIALS AND METHODS 2.1. Chemicals. A mixture of carbonic anhydrase (EC.4.2.1.1) isoforms from bovine erythrocytes, 4-nitrophenyl acetate (NPA), sodium phosphate, glutaraldehyde (GA), aniline, 3-aminobenzoic acid (3-ABA), ethanol, acetonitrile, ammonium persulfate, ammonium sulfate (AS), hydrochloric acid (HCl), and Tris-HCl were purchased from Sigma (St Louis, MO, USA). 1-Ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 2-(N-morpholino) ethanesulfonic acid (MES) were purchased from Pierce (Rockford, IL, USA). Amine functionalized magnetic nanoparticles (MNPs) were purchased from Chemicell (Berlin, Germany). 2.2. Polymerization of Aniline and 3-Aminobenzoic Acid. The copolymerization of aniline and 3-aminobenzoic acid (3-ABA) was previously reported16,17 and a modified protocol was carried out. Carboxylated polyaniline nanofibers (cPANFs) were synthesized by varying the molar ratio of aniline and 3ABA. Various molar ratios of aniline and 3-ABA were mixed with HCl (1 M), and shaken (200 rpm) at 50 °C for 1 h. Then, the same volume of 0.1 M ammonium persulfate solution in HCl (1 M) was added to initiate the polymerization reaction at room temperature under shaking (200 rpm) for 24 h. After polymerization, the samples were washed with distilled water three times. Finally, the mass of polymers was measured after vacuum drying at 50 °C for 17 h, and the polymers were characterized by Fourier transform infrared (FTIR) spectroscopy (Spectrum GX, PerkinElmer, Waltham, MA, USA).
Figure 2. Schematic illustrations for immobilization of carbonic anhydrase on cPANF. B
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Figure 3. SEM and HR-TEM images. (a) SEM images and (b) HR-TEM images of pristine cPANF, immobilized carbonic anhydrases on cPANFs via four different approaches, which are CA, EC, EPC, and Mag-EPC. Black dots in the HR-TEM images of Mag-EPC represent magnetic nanoparticles incorporated with carbonic anhydrases on cPANFs.
2.5. Activity and Stability Measurements. The enzyme activity was measured by the hydrolysis of 4-nitrophenyl acetate (NPA) in aqueous buffer (100 mM PB, pH 7.6).18 A 60 mM NPA (50 μL) aliquot was mixed with PB (850 μL) in a cuvette, and a 100 μL enzyme sample was added to initiate the enzyme reaction. The increase of absorbance at 348 nm was measured by using a spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan). To check the enzyme stability, free carbonic anhydrase, CA, EC, EPC, and Mag-EPC were incubated in 100 mM PB (pH 7.6) at room temperature under shaking (200 rpm). At each time point, an aliquot was removed from the stock sample, and used for the activity measurement. The relative activity was calculated from the ratio of residual activity at each time point to the initial activity of each enzyme sample, and used to prepare the stability curve of each sample. The stability of Mag-EPC in 70% ethanol was also checked by measuring the residual activity time-dependently after incubation in 70% ethanol at room temperature under shaking (200 rpm). At each time point, an aliquot from the stock was excessively washed via three cycles of magnetic capture and washing with 100 mM PB (pH 7.6), and used for the activity measurement. 2.6. Microalgal Cultivation with Carbonic Anhydrase on Carboxylated Polyaniline Nanofiber. Dunaliella tertiolecta (D. tertiolecta) ATCC 30929 was cultivated in a 100 mL culture flask, containing 20 mL of modified artificial seawater D medium, which is composed of 40 mM Tris-HCl (pH 7.4),19,20 1.0 M NaCl, 5 mM KNO3, 4.5 mM MgCl2·6H2O, 0.5 mM MgSO4·7H2O, 0.3 mM CaCl2, 0.1 mM K2HPO4, 2 μM FeCl3, 20 μM EDTA, 50 μM H3BO3, 10 μM MnCl2·4H2O, 0.8 μM ZnSO4·7H2O, 0.4 μM CuSO4·5H2O, 2 μM NaMoO4·2H2O, 0.2 μM CoCl2·6H2O, and 1.5 μM NaVO3. A 1 mL aliquot of Mag-EPC (1 mg cPANF/mL) was sterilized in 70% (v/v) ethanol for 30 min. Then, the Mag-EPC was washed three times with autoclaved 40 mM Tris buffer (pH 7.4), and added to the culture of D. tertiolecta. D. tertiolecta cultivations were carried out at 23 °C under illumination with white fluorescent light (50 μmol photons m−2 s−1) on a tilt shaker at a speed of 26 rpm. The initial cell concentration was adjusted to 50 × 104 cell/mL. For the case of Mag-EPC, 1 mL of Mag-EPC was added to the culture medium. For the controls, Mag-cPANF was added instead of Mag-EPC, while in the case of cell only, cells were cultivated with neither Mag-EPC nor Mag-cPANF
anhydrase. Carboxyl groups of cPANF were modified by EDC/ NHS for covalent attachment of carbonic anhydrase. cPANF (2 mg) was mixed with EDC (10 mg/mL) and NHS (50 mg/mL) in MES buffer (100 mM, pH 6.0), and the mixture was shaken at 50 rpm for 1 h. EDC/NHS-treated cPANF was washed by distilled water two times, and recovered via centrifugation. The carbonic anhydrase solution (1 mL) was mixed with EDC/ NHS-treated cPANF, and the mixture was incubated at room temperature under shaking at 150 rpm for 2 h for the covalent attachment of enzyme molecules onto cPANFs. Aminefunctionalized magnetic nanoparticles (MNP) and ammonium sulfate were added, and the mixture was shaken (150 rpm) for 30 min. Then, the GA solution was added to the mixture to make the final concentration of 0.5% (w/v) GA, and the mixture was incubated at room temperature under shaking at 50 rpm for 30 min. For the preparation of the CA sample, ammonium sulfate, GA solution, and MNP solution were replaced with 100 mM PB (pH 7.6). The EC sample did not use ammonium sulfate and MNP while EPC did not use MNP. For Mag-cPANF, carbonic anhydrase was not added. All the samples were incubated at 4 °C under shaking at 50 rpm for 17 h. After washing three times by 100 mM PB (pH 7.6), the mixtures were incubated in 100 mM Tris-HCl buffer (pH 7.6) under shaking (200 rpm) for 30 min in order to cap the unreacted aldehyde groups. The samples were washed three times with 100 mM PB (pH 7.6) under shaking (250 rpm), followed by centrifugation at 10 000 rpm (in the cases of CA, EC, and EPC) or by magnet capture for 2 min (in the case of Mag-EPC) to recover the samples. Finally, CA, EC, EPC, and Mag-EPC were stored in 100 mM PB (pH 7.6) at 4 °C until use. 2.4. The Scanning Electron Microscope (SEM) and High Resolution-Transmission Electron Microscope (HRTEM) Analyses. cPANFs, CA, EC, EPC, and Mag-EPC were analyzed by using a SEM (S-4800, Hitachi, Tokyo, Japan) and the HR-TEM (TECNAI G2 F30 ST, FEI, Hillsboro, OR, USA). For the SEM analysis, all the samples were washed by distilled water and freeze-dried for 24 h. After platinum (Pt) coating, the samples were imaged at an accelerating voltage of 15 kV. For the HR-TEM analysis, all the samples were washed by distilled water, and 5 μL of washed samples were dropped on the copper (Cu) grid, followed by drying at room temperature for 24 h. The dried samples were imaged at an accelerating voltage of 200 kV. C
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buffer (PB, pH 7.6). The measured activities of CA, EC, EPC, and Mag-EPC were 0.18 ± 0.01, 0.061 ± 0.01, 6.4 ± 0.1, and 9.2 ± 0.2 units per milligram of cPANFs, respectively (Figure 4). One unit is defined by the enzyme amount to hydrolyze 1
addition. After 6 days of cultivation, the cell number was counted using a microscope (CH30, Olympus, Tokyo, Japan). For the recycling of Mag-EPC, Mag-EPC was captured by a magnet for 5 min after 6 days of cultivation, and sterilized in 70% (v/v) ethanol for 30 min. Then, Mag-EPC was washed three times with autoclaved 40 mM Tris buffer (pH 7.4), and added to the newly prepared culture of D. tertiolecta. Cultivations were carried out at 23 °C under illumination with white fluorescent light (50 μmol photons m−2 s−1) on a rocker at a speed of 26 rpm.
3. RESULTS AND DISCUSSION 3.1. Immobilization and Stabilization of Carbonic Anhydrase on Carboxylated Polyaniline Nanofibers. In the present work, we report, for the first time, the synthesis of carboxylated polyaniline nanofibers (cPANFs), which can be produced in a large-scale and economical way. Copolymerization of aniline and 3-aminobenzoic acid (3-ABA) was performed to prepare cPANFs (Figure S1 and S2 in Supporting Information), which have a nanofiber matrix with porous interfiber space and provide carboxyl groups for covalent attachment of enzymes. Carbonic anhydrases were immobilized on cPANFs using four different approaches, such as covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetically separable EPC (Mag-EPC) (Figure 2). CA was prepared by forming covalent bonds between enzymes and cPANFs using 1-ethyl-3-(3(dimethylamino)propyl) carbodiimide (EDC) linker, while the enzyme cross-linking was additionally performed via glutaraldehyde (GA) treatment to prepare the EC sample. EPC was prepared via a three-step process, consisting of covalent attachment, enzyme precipitation, and cross-linking, where ammonium sulfate was added for the precipitation of enzymes. For Mag-EPC, amine-functionalized magnetic nanoparticles were mixed with carbonic anhydrase in the beginning step of EPC synthesis. Figure 3 shows the scanning electron microscope (SEM) and high resolution-transmission electron microscope (HR-TEM) images of pristine cPANF, CA, EC, EPC, and Mag-EPC. CA and EC showed negligible change in the morphology and thickness of nanofibrous structures when compared to pristine cPANFs, whereas EPC and Mag-EPC showed a significant increase in the thickness of nanofibrous structures. According to the SEM images, the average thicknesses of cPANF, CA, EC, EPC and Mag-EPC were estimated to be 52 ± 10, 54 ± 8, 59 ± 9, 116 ± 18, and 120 ± 19 nm, respectively. The analysis of the HR-TEM images resulted in the estimation of 47 ± 12, 48 ± 10, 50 ± 7, 105 ± 14, and 108 ± 18 nm, respectively, showing a similar trend to the estimation from the SEM images. These results suggest that a larger amount of carbonic anhydrase was immobilized in the form of EPC by a simple addition of enzyme precipitation step before the enzyme cross-linking step.21 The HR-TEM image of Mag-EPC shows magnetic nanoparticles all over the nanofibrous structures, revealing the effective cross-linking of amine-functionalized magnetic nanoparticles together with carbonic anhydrase on cPANFs. The SEM analysis of each sample resulted in a marginally increased average thickness than the TEM analysis, which can be explained by the Pt coating for the SEM analysis. 3.2. Activity and Stability of Carbonic Anhydrase on Carboxylated Polyaniline Nanofibers. The activities of CA, EC, EPC, and Mag-EPC were measured by the hydrolysis of 4nitrophenyl acetate (NPA) in 100 mM sodium phosphate
Figure 4. Activities of CA, EC, EPC, and Mag-EPC in the hydrolysis of 4-nirophenyl acetate. One unit is defined by the enzyme amount to hydrolyze 1 μmole NPA per 1 min in 100 mM sodium phosphate buffer (pH 7.6).
μmole NPA per 1 min in 100 mM sodium phosphate buffer (pH 7.6). EC having lower activity than CA can be explained by two reasons, the enzyme inactivation during the glutaraldehyde treatment and the reduced structural flexibility of enzymes upon cross-linking. Higher activities of EPC and Mag-EPC than those of CA and EC can be explained by higher loading of carbonic anhydrase on cPANFs, as observed in SEM and TEM images (Figure 3). Interestingly, the activity of Mag-EPC was 1.4 times higher than that of EPC. This result can be explained by magnetic nanoparticles with a lot more amine groups than carbonic anhydrase, which can potentially improve the efficiency of cross-linking and result in higher enzyme loadings. Unfortunately, it is hard to measure the enzyme loading due to the interference of protein assay by the presence of insoluble forms of cross-linked enzymes and amine-functionalized magnetic nanoparticles.22 We have also measured the activities of CA, EC, EPC, and Mag-EPC via the Wilbur−Anderson (WA) assay,23 which performs the conversion of CO2 to bicarbonate and measures the time for the buffer pH drop from 8.3 to 6.3. Even though the activities obtained via the W-A assay cannot be directly compared to those from the NPA assay, the comparison of immobilized enzymes in their activities showed a similar trend regardless of which enzyme assay was used (Supporting Information, Table S1). The stabilities of free carbonic anhydrase, CA, EC, EPC, and Mag-EPC were investigated by measuring the enzyme activity time-dependently after incubating the samples in aqueous buffer (100 mM PB, pH 7.6) at room temperature under shaking (200 rpm). Free carbonic anhydrase, CA and EC showed a monotonous decrease of enzyme activity with the half-lives of 3.0, 7.1, and 13.5 days, respectively. On the other hand, EPC and Mag-EPC showed a good stabilization of enzyme activity, and their half-lives were 180 and 236 days, respectively (Figure 5a). When considering the data points after 150-day incubation, the half-lives of EPC and Mag-EPC were calculated to be 367 and 686 days, respectively. Upon chemical cross-linking, the resulting cross-linked enzymes can be divided into two different populations: stable and labile forms. The stable form would represent the population of cross-linked D
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separation but also improved both enzyme loading and stability due to more amino groups on the surface of magnetic nanoparticles that can create more chemical cross-linkages. The use of cPANFs must contribute to the improvement of enzyme loading and stability because the carboxyl groups of cPANFs allow for the chemical linkages between cPANFs and crosslinked enzymes by using the EDC linkers. We further investigated the stability of Mag-EPC in 70% (v/ v) ethanol solution at room temperature under shaking (200 rpm) (Figure 5b). After 300 min incubation, Mag-EPC showed 92 ± 4% of its initial activity, and the half-life was calculated to be 45.2 h. This result suggests that the cross-linking of enzyme molecules in the form of Mag-EPC can place a strong resistance against enzyme denaturation even in 70% (v/v) ethanol solution. This feature of Mag-EPC allows for its facile option of sterilization by using 70% (v/v) ethanol, which is critical for the success of one-pot carbon conversion and utilization in the form of improved microbial growth. 3.3. Microalgal Cultivation with Stabilized and Magnetically Separable Carbonic Anhydrase. We have checked if Mag-EPC can be effectively employed for one-pot CO2 conversion and utilization by directly putting the ethanolsterilized Mag-EPC into the culture of photosynthetic organisms. Photosynthetic organisms have been genetically modified to improve their ability to fix CO2 and biomass productivity.26−31 Despite these efforts, the cultivation of microalgae in open pond is still slow because of low CO2 concentration, placing a bottleneck against the large-scale and productive cultivation. As a potential solution, Mag-EPC of carbonic anhydrase can increase the concentration of inorganic carbon in the form of bicarbonate for the expedited growth of photosynthetic organisms. To develop a protocol of one-pot CO2 conversion and utilization, ethanol-sterilized Mag-EPC was added to the culture of D. tertiolecta, a model microalga, which can predominantly uptake HCO3− compared to CO2 in aqueous solution.32 This microalga is known to accumulate a high amount lipids (up to 34% of its dry weight), and has a relatively high growth rate that makes it a good biocatalyst for the production of biodiesels and other value-added chemicals.33,34 D. tertiolecta was cultivated in the presence of Mag-EPC with no additional inorganic carbon source under atmospheric CO2 (0.04%) in modified artificial seawater D medium (pH 7.4). Two different control samples were prepared for comparative studies: cells only and MagcPANF (only magnetic particles on cPANFs with no carbonic anhydrase). After cultivation for 6 days, the average cell concentrations of cell only and Mag-cPANF were 433 ± 21 × 104 cells/mL and 430 ± 52 × 104 cells/mL, respectively, while the addition of Mag-EPC resulted in the cell concentration of 756 ± 23 × 104 cells/mL, representing 1.8-fold increase when compared to the control samples (Figure 6a). This implies that Mag-EPC successfully converted CO2 into HCO3− and the cell growth was improved by consuming HCO3− as an inorganic carbon source. We have further checked the cell growth in the presence of free carbonic anhydrase with the same NPA activity to Mag-EPC, and found that free carbonic anhydrase resulted in 6% increase of cell number when compared to that of MagEPC (Supporting Information, Figure S4). This marginally increased cell number with free carbonic anhydrase can be explained by improved interfacial biocatalysis transforming CO2 from atmosphere to bicarbonate into microalgae culture because they are soluble and better exposed to atmosphere for interfacial biocatalysis.
Figure 5. Stabilities of carbonic anhydrase samples; (a) stability of free carbonic anhydrase, CA, EC, EPC, and Mag-EPC under shaking (200 rpm); (b) stability of Mag-EPC in 70% (v/v) ethanol.
enzymes with a greater number of chemical cross-linking, while the labile form would represent with a smaller number of chemical cross-linking. The biphasic inactivation kinetics of EPC and Mag-EPC can be explained by the completion of labile form inactivation up to 150 days, followed by the sole exposure of inactivation of the still-alive stable form. Because of more chemical linkages and improved stability, the half-lives of stable forms were improved when calculated at the later phase after 150 days. We have also checked the stabilities of free carbonic anhydrase, CA, EC, EPC, and Mag-EPC via the W-A assay, and compared them with the stability results measured by the NPA assay (Supporting Information, Figure S3). Even though CA and EC samples showed a little bit of different trends depending on which assay to be used, the overall trends in the stabilities of immobilized enzymes from the standpoint of comparative studies were similar regardless of which enzyme assay was used. The improved stability of EPC and Mag-EPC can be explained by the multipoint covalent linkages that can effectively prevent both denaturation and leaching of enzymes.24,25 In other words, the multiple chemical linkages on the surface of enzymes can efficiently inhibit not only the unfolding of each enzyme molecule but also the detachment of enzyme molecules. The comparison of loadings and stabilities between EC and EPC suggest that the step of enzyme precipitation is critical for the efficient formation of chemical cross-linkages during the follow-up enzyme cross-linking. This result can be explained by the shortened distances among enzymes and cPANFs when enzyme molecules are precipitated. The addition of amine-functionalized magnetic nanoparticles for Mag-EPC has not only enabled the facile magnetic E
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utilization based on Mag-EPC, first reported in this study, can be employed not only to accelerate the growth of various microalgae that can produce useful chemicals, but also to combine with various other enzymes using bicarbonate as a substrate for one-pot CO2 conversion and utilization under the concept of substrate channeling.35 Further, the approach of Mag-EPC can be employed for other enzyme applications such as biosensors, biofuel cells, and enzymatic biodiesel production, where the poor enzyme stability hampers their practical applications.
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ASSOCIATED CONTENT
S Supporting Information *
Activities and stabilities of CA, EC, EPC, and Mag-EPC measured by 4-nitrophenyl acetate (NPA) and WilburAnderson (W-A) assays; FTIR spectra; synthesis of carboxylated polyaniline nanofibers (cPANFs); growth of D. tertiolecta in the presence of Mag-EPC and free carbonic anhydrase. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*Fax: +82-2-925-4850; e-mail:
[email protected]. *Fax: +82-2-2299-2561; e-mail:
[email protected]. Author Contributions #
S.-G.H. and H.J. contributed equally to this work.
Notes
Figure 6. Microalgal production in one-pot conversion and utilization of CO2. (a) Growth of D. tertiolecta in the presence of Mag-EPC. (b) Repetitive uses of Mag-EPC for the growth of D. tertiolecta. Cell concentration in the first cycle after 6 days of cultivation was 740 × 104 cells/mL.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by grants from the International Collaborative R&D Program (No. 20118510020020) and Energy Efficiency & Resources Core Technology Program (No. 20142020200980) of the Korea Institute of Energy Technology Evaluation and Planning (KETEP), funded by the Korea government Ministry of Trade, Industry & Energy. This work was also supported by the Korea CCS R&D Center (KCRC) (NRF-2014M1A8A1049273), by the Advanced Biomass R&D Centre (ABC) of Global Frontier Project (ABC-M3A6A2079376), by the Nano·Material Technology Development Program (2014M3A7B4052193), and by the Global Research Laboratory Program (2014K1A1A2043032), all of which are funded by the Korea government Ministry of Science, ICT & Future Planning.
We further investigated the recycled uses of Mag-EPC by using its facile magnetic separation. Especially, the good stability of Mag-EPC in 70% (v/v) ethanol solution allows for an easy sterilization of Mag-EPC before each recycled use in the culture of microalgae. Mag-EPC was magnetically captured within 5 min, sterilized in 70% (v/v) ethanol for 30 min, and reused for the improved growth of D. tertiolecta. Under repetitive uses for expedited D. tertiolecta cultivation (6 days of cultivation per each cycle), the average cell number after each cycle was counted, and the relative cell number was calculated by the ratio of average cell number of each cycle to that of the first cycle. Relative cell number was maintained by reusing MagEPC (Figure 6b), suggesting that the cell growth was unaffected by the repeated use of Mag-EPC. These results indicate that highly stable Mag-EPC can be fully recovered within 5 min of magnet capture and maintain its activity under the repetitive cultivation and 70% (v/v) ethanol sterilization three times. Magnetically separable and highly stable Mag-EPC can be successfully recycled to make a bicarbonate-rich condition by capturing atmospheric CO2 and converting it to bicarbonate simultaneously, enabling the successful one-pot CO2 conversion and utilization. In summary, carbonic anhydrase was successfully immobilized and stabilized via the Mag-EPC approach for one-pot CO2 capture and utilization to accelerate the growth of microalgae, D. tertiolecta. The addition of Mag-EPC converts CO2 into bicarbonate in the culture medium, which enables the accelerated growth of D. tertiolecta without an additional inorganic carbon source. The one-pot CO2 conversion and
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REFERENCES
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DOI: 10.1021/es505143f Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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DOI: 10.1021/es505143f Environ. Sci. Technol. XXXX, XXX, XXX−XXX
