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Biomimetic CO2 sequestration using purified carbonic anhydrase from indigenous bacterial strains immobilized on biopolymeric materials
Enzyme and Microbial Technology 48 (2011) 416–426
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Biomimetic CO2 sequestration using purified carbonic anhydrase from indigenous bacterial strains immobilized on biopolymeric materials Anjana Sharma ∗ , Abhishek Bhattacharya, Ankita Shrivastava Bacteriology Laboratory, Department of P.G. Studies and Research in Biological Science, Rani Durgavati University, Pachpedi, Jabalpur 482001, Madhya Pradesh, India
a r t i c l e
i n f o
Article history: Received 3 October 2010 Received in revised form 2 February 2011 Accepted 2 February 2011 Keywords: Biomimetic CO2 -sequestration Immobilization Carbonic anhydrase P. fragi Indigenous
a b s t r a c t The present study deals with immobilization of purified CA and whole cell of Pseudomonas fragi, Micrococcus lylae, and Micrococcus luteus 2 on different biopolymer matrices. Highest enzyme immobilization was achieved with P. fragi CA (89%) on chitosan–KOH beads, while maximum cell immobilization was achieved with M. lylae (75%) on chitosan–NH4 OH beads. A maximum increase of 1.08–1.18 fold stability between 35 and 55 ◦ C was observed for M. lylae immobilized CA. The storage stability was improved by 2.02 folds after immobilization. FTIR spectra confirmed the adsorption of CA on chitosan–KOH beads following hydrophilic interactions. Calcium carbonate precipitation was achieved using chitosan–KOH immobilized P. fragi CA. More than 2 fold increase in sequestration potential was observed for immobilized system as compared to free enzyme. XRD spectra revealed calcite as the dominant phase in biomimetically produced calcium carbonate. © 2011 Elsevier Inc. All rights reserved.
1. Introduction The rising concentration of green house gases (GHGs), and in particular carbon dioxide (CO2 ) due to anthropogenic interventions has led to several undesirable consequences such as global warming and related changes. The most easily addressable source of CO2 is that from the generation of electric power, and particularly that from the coal burning stations, since they comprise a relatively small number of very large stationary sources [1]. Coal fired power plants currently account for little more than half the electricity generation and it is unlikely that there will be a dramatic change in this situation in the next twenty years [2,3]. Conversion of CO2 into solid carbonates offers the possibility of a safe and stable ecofriendly product for long term carbon sequestration [2]. Precipitation from aqueous solution occurs at a suitable supersaturation of calcium and carbonate ions [4]. The hydration of CO2 to form carbonic acid is the rate-limiting step in the conversion of CO2 into carbonate ions, which has a forward reaction constant of 6.2 × 10−3 s at 25 ◦ C [5,6]. This hydration reaction is catalyzed by carbonic anhydrase (CA) at or near diffusion controlled limit [7]. Carbonic anhydrase (CA) is one of the fastest enzymes that catalyses CO2 -hydration reaction with typical rates between 104 and 106 reactions per second for different forms of this enzyme [8]. It is a zinc metalloenzyme reported to be present in animals, plants and
microorganisms [9–12]. The successful precipitation of CO2 (aq) into CaCO3 in the presence of calcium ions through biomimetic approach involving, indigenous carbonic anhydrase [13] and commercial Bovine Carbonic anhydrase (BCA) [14] has been proved in principle. However, commercial application of this approach warrants the immobilization of CA on to a suitable immobilization matrix for application at an onsite scrubber [14]. The process of immobilization confines enzymes/whole cells to a phase distinct from the one in which the substrates and the products are present [15]. Chitosan and sodium alginate are inert materials that have been used for immobilization of enzymes and microorganism [15]. Immobilization enhances the efficiency of the process by allowing reuse of enzyme coupled with easy removal of the products from the reaction mixture. The physical methods especially adsorption, have an advantage over the chemical methods for immobilization of enzymes onto carriers in that it is simple, less expensive and can retain high catalytic activity [16]. CO2 sequestration using immobilized BCA and indigenous CA under different process parameters is under research phase [13,15] and an objectively defined study involving indigenous bacterial CA immobilized on inert matrix is the need of the hour. The present study thus aims at immobilization of both CA and bacterial cells of indigenous origin on chitosan and alginate based materials and to assess their CO2 sequestration potential. 2. Materials and methods
∗ Corresponding author. Tel.: +91 761 2416667; fax: +91 761 2603752; mobile: +91 9425155323. E-mail address: [email protected] (A. Sharma). 0141-0229/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2011.02.001
Artificial sea water (ASW) and chitosan were purchased from Sigma–Aldrich, St Louis, MO, USA. Sodium alginate was purchased from Himedia Mumbai, India. All the buffers used in the study were purchased from Himedia Mumbai, India. The sodium
A. Sharma et al. / Enzyme and Microbial Technology 48 (2011) 416–426 and potassium salts of chloride, bromide, iodide, fluoride, carbonate, sulphate and nitrate were purchased from Sisco Research Laboratory, Mumbai, India.
2.1. Synthesis of materials Chitosan–NaOH beads (B-1), chitosan–KOH beads (B-2), chitosan–NH4 OH beads (B-3), sodium alginate–CaCl2 beads (B-4), chitosan–sodium alginate–CaCl2 beads (B-5) and multilayered beads (B-6), were prepared following the method of Prabhu et al. [15].
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2.5. Effect of pH and temperature on immobilized CA The stability of the CA immobilized on chitosan–KOH beads was assayed using two different buffer systems, 50 mM phosphate buffer (pH 7.0, 7.5 and 8.0) and Tris–HCl buffer (pH 8.5 and 9.0) at 37 ◦ C for 3 h. The temperature stability of immobilized CA on chitosan–KOH beads was determined by carrying out the enzyme reactions at different temperatures (35–55 ◦ C) and pH 8.0 for 3 h. The enzyme activity at the start of the experiment was taken as 100% and the residual activity was determined after incubation. 2.6. Storage stability
2.2. Source of the bacterial strain and carbonic anhydrase The indigenous purified CA from Pseudomonas fragi (BGCC = / 1077), Micrococcus lylae (BGCC = / 1078), and Micrococcus luteus 2 (BGCC = / 1079) were obtained from Bacterial Germplasm Collection Centre (BGCC), Bacteriology Laboratory, Department of P.G. Studies and Research in Biological Science, R.D. University, Jabalpur (M.P.), India.
2.3. Immobilization 2.3.1. Immobilization of enzyme Lyophilized CA (5 mg) was dissolved in 5 ml of 50 mM Tris–HCl (pH 8.0) and mixed separately with 1 g of the immobilization matrix. The experimental setup containing 3 sets was designed. Each set with five separate tubes contained enzyme immobilization matrix suspension for P. fragi CA, M. lylae CA and M. luteus 2 CA, separately. The suspension in all the three sets was agitated (120 rpm) at 4 ◦ C and the immobilization efficiency was determined at every 2 h interval up to 10th h, using a single tube containing the suspension each from the three sets designated for CA from the three indigenous strains. After immobilization the beads were separated from the suspension and enzyme activity (EU) and protein concentration (mg) in the supernatant was determined. The direct estimation of CO2 hydration activity on beads after immobilization was carried out using 200 mg immobilized enzyme. The total enzyme activity on the beads was also determined as the difference between the total enzyme activity of free enzyme in solution before immobilization and the total enzyme activity present in the solution after immobilization. The total enzyme activity of immobilized CA was expressed as EU/g beads and the mass transfer of enzyme on immobilized beads was determined as amount of protein retained on beads after immobilization i.e. difference in protein concentration before and after immobilization in the suspension and was expressed as g protein/g beads. The specific activity (U) [where, U = EU/mg protein], on the immobilized beads was also determined and was expressed as U/g beads. The immobilization potential (%) was determined in terms of specific activity retained on beads compared to that of free enzyme (100%).
2.3.2. Immobilization of microorganisms Culture (100 ml) of each strain (A600 nm = 1.2) and 1 g of different immobilization matrices was added into flask and incubated at 37 ◦ C for 10 h with 2 h interval at 120 rpm. After incubation the slurry was centrifuged and the beads were collected, washed thoroughly with sterile distilled water and suspended in Tris–HCl buffer (50 mM, pH 8.0). The sample was sonicated thrice for 10 s with 30 s intervals followed by centrifugation at 10,000 rpm for 10 min [15]. Similarly the culture (A600 nm = 1.2) without any immobilization matrix was centrifuged and suspended in Tris–HCl buffer (50 mM, pH 8.0), followed by sonication and centrifugation as described above. The supernatant obtained was used to determine the enzyme activity (EU) and protein concentration (mg). The total enzyme activity of immobilized cells was expressed as EU/g beads and the mass transfer of cells on immobilized beads was determined as amount of protein retained on beads after immobilization and was expressed as g protein/g beads. The specific activity (U) [where, U = EU/mg protein], on the immobilized beads was also determined and was expressed as U/g beads. The immobilization potential (%) was determined in terms of specific activity retained on beads compared to that of free culture (100%).
2.4. Determination of carbonic anhydrase activity The method of Sharma and Bhattacharya [13] was followed with certain modifications. In brief, carbon dioxide saturated water prepared by introducing CO2 (100 kPa) in 500 ml of Milli Q grade pure water for 1 h at 4 ◦ C. CO2 saturated water (3 ml) was immediately added to 2 ml of Tris–HCl buffer (100 mM; pH 8.3) [13], and 0.1 ml of free purified CA (1 mg/ml stock), 0.1 ml of supernatant left after the immobilized enzyme was separated from suspension and 0.1 ml of supernatant derived after sonication of cell culture and immobilized cells were transferred immediately but separately to determine the enzyme activity. The time required for the pH change 8.0–7.0 (t) was measured. The time required for the pH change (8.0–7.0) was used as control (tc ), when buffer was substituted for test sample. The enzyme assay was carried out at 4 ◦ C. The Wilbur Anderson units were calculated with the equation (tc − t)/t [17]. The protein content was determined by the method of Lowry et al. [18].
The storage stability of both the free enzyme and immobilized enzyme was determined by storing them for 30 days at 4 ◦ C. The residual activity was determined following the standard enzyme assay method. 2.7. Characterization of the immobilization matrix and immobilized enzyme FTIR spectra of chitosan flakes, chitosan–KOH beads, free carbonic anhydrase and CA immobilized on chitosan–KOH beads mixed with KBr pellets were recorded on Shimadzu FTIR spectrophotometer (V-110). Spectra of all the materials were scanned in the range of 400–4000 cm−1 . 2.8. Determining CO2 sequestration efficiency using immobilized carbonic anhydrase CO2 saturated solution using artificial sea water (ASW) was prepared at room temperature as described under Section 2.4. ASW was supplemented with K2 SO4 (0.1 M) and KNO3 (0.1 M) to study the effect of SOx and NOx on CA activity and sequestration efficiency. CO2 saturated solution (10 ml) was mixed with 1 ml of Tris buffer (pH 8.3) containing free CA (100 g) from all three bacterial strains separately for 15 min. The bicarbonate solution was released into another vessel through a valve containing 10 ml of CaCl2 solution (at a final concentration of 10.0 mM). To the above mixture, Tris-buffer pH 9.5 (2 ml, 1 M) was immediately added. The reaction mixture was incubated at 35 ◦ C and 45 ◦ C, respectively for 5 min to allow precipitation of CaCO3 and the amount of CaCO3 formed was determined [13]. The control experiment was carried out in the absence of enzyme and the results were expressed in terms of mg CaCO3 formed following control correction. CA from all the three indigenous isolates immobilized on chitosan–KOH beads was used for sequestration studies by replacing the free enzyme in the reaction chamber. The sequestration efficiency was evaluated by determining the ionic concentration of calcium present before and after carbonate precipitation following the method of Kolthof et al. [19]. The difference in calcium concentration was considered to be the amount of calcium utilized in formation of calcium carbonate. An enzyme free system was used as control for comparison. The percentage efficiency of calcium ion utilized was also calculated and results were reported following control correction. The weight of calcium carbonate was also calculated and gm equivalents of CO2 present in CaCO3 were also determined. 2.9. Reusability of immobilized CA for CO2 sequestration The immobilized CA was evaluated for reusability in 10 batch process. The reaction of CO2 saturated solution with immobilized enzyme was carried out in a chamber separate from the vessel used for CaCO3 precipitation. The CA beads were rinsed with distilled water to neutralize the pH and to remove any excess ions. The chamber is separated from the CO2 reservoir and precipitation vessel being connected through a two way valve allows its easy removal, washing of beads and refining into the main apparatus for further reuse. 2.10. XRD analysis The X-ray diffraction spectra of biomimetically produced CaCO3 were recorded on a Rigaku Miniflex II instrument using Cu K␣ radiation ( = 0.15406 nm) operated at 30 kV and 15 mA. 2.11. SEM analysis Scanning electron microscopy was performed on selected carbonate samples generated biomimetically. A JEOL JSM 5800 LV electron microscope under an electrical tension of 20 kV was used for this purpose. A small solid piece of each sample to be examined was placed on a sample holder covered with a carbon tab and metalized with gold for 2.5 min in a cathodic atomizer blazer (20).
3. Results The specific activity of purified CA (1 mg/ml) from P. fragi (70.6 U), M. lylae (66.5 U) and M. luteus 2 (61.0 U) was determined following CO2 hydration assay.
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Table 1 Comparison of enzyme activity (EU/g of beads), protein content (g/g beads), specific activity (U/g) and immobilization potential (%) of purified P. fragi carbonic anhydrase. Immobilization period
Immobilization matrix Chitosan–NaOH (B-1)
2h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 4h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 6h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 8h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 10 h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g)
Chitosan–KOH (B-2)
Chitosan–NH4 OH (B-3)
Sodium alginate–CaCl2 (B-4)
Chitosan alginate–CaCl2 (B-5)
Multilayered beads (B-6)
46.98 0.0018 26.1 (37)
62.16 0.0021 29.6 (42)
56.4 0.002 28.2 (40)
12.06 0.0009 13.4 (19)
13.90 0.0001 13.9 (20)
11.7 0.0009 13 (18)
88.25 0.0025 35.3 (50)
120.64 0.0029 41.6 (59)
102.87 0.0027 38.1 (54)
24.83 0.0013 19.1 (27)
28.56 0.0014 20.4 (29)
28 0.0014 20 (28)
175.35 0.0035 50.1 (71)
214.89 0.0039 55.1 (78)
192.4 0.0037 52.0 (75)
31.2 0.0016 19.5 (32)
52.25 0.0019 27.5 (39)
33.0 0.0015 22 (31)
229.2 0.0004 57.3 (80)
276.32 0.0044 62.8 (89)
249.06 0.0042 59.3 (84)
50.92 0.0019 26.8 (38)
62.16 0.0021 29.6 (42)
60.9 0.0021 29 (41)
234.52 0.0041 57.2 (81)
276.32 0.0044 62.8 (89)
249.06 0.0042 59.3 (84)
50.92 0.0019 26.8 (38)
62.16 0.0021 29.6 (42)
42.5 0.0017 25 (35)
The enzyme activity, protein content and specific activity for free purified enzyme were 353 (EU), 0.005 (g) and 70.6 U (100%) respectively. The values within the parentheses indicate the % immobilization. All the errors were within 5% of SD.
3.1. Screening of materials for immobilization of enzyme and cells Determination of enzyme and cell immobilization in terms of specific activity showed that maximum CA immobilization was achieved after 8 h of incubation; while maximum cell immobilization was achieved following 10 h of incubation irrespective of the type of immobilization material used. Maximum immobilization of CA from all the three strains was obtained on B-2 followed by B-3 and B-1, less than 50% immobilization was achieved with B-4, B5 and B-6 materials (Table 1, Table 2, and Table 3). CA immobilized on chitosan–KOH (B-2) was used for further
studies. The immobilization potential (%), enzyme activity (EU/g beads) and mass transfer (g/g beads) were determined for CA from all the three strains (Table 1, Table 2, and Table 3). Maximum mass transfer (g/g beads) and enzyme activity (EU/g beads) was observed on chitosan–KOH (B-2) beads for CA from P. fragi (0.0044 g/g beads; 276.32 EU/g beads) followed by M. lylae (0.0042 g/g beads; 231.84 EU/g beads) and M. luteus 2 (0.0042 g/g beads; 215.04 EU/g beads). Maximum cell immobilization for P. fragi was observed on B-6, followed by B-4 and B-5. Minimum immobilization was observed on B-2. However, maximum cell immobilization for M. lylae and M. luteus 2 was observed on B-3 followed by B-1 and B-2. Min-
Table 1a Comparison of enzyme activity (EU/g of beads), protein content (g/g beads), specific activity (U/g) and immobilization potential (%) of whole cell P. fragi (carbonic anhydrase). Immobilization period
2h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 4h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 6h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 8h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 10 h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g)
Immobilization matrix Chitosan–NaOH (B-1)
Chitosan–KOH (B-2)
Chitosan–NH4 OH (B-3)
1.42 0.0016 0.89 (15)
0.19 0.0006 0.32 (5.3)
0.36 0.0008 0.45 (7.3)
3.17 0.0023 1.38 (22)
3.36 0.0024 1.40 (23)
3.40 0.0024 1.42 (23)
2.58 0.0021 1.23 (20)
0.40 0.0008 0.48 (7.8)
0.57 0.0010 0.57 (9.3)
3.70 0.0025 1.48 (24)
3.28 0.0024 1.37 (22)
4.41 0.0027 1.63 (26)
3.10 0.0023 1.35 (22)
1.04 0.0013 0.80 (13)
1.32 0.0015 0.88 (14)
9.28 0.0040 2.32 (38)
6.73 0.0034 1.98 (32)
9.76 0.0041 2.38 (39)
3.13 0.0023 1.36 (22)
1.35 0.0015 0.90 (14)
1.7 0.0017 1.00 (16)
11.79 0.0045 2.62 (43)
6.73 0.0034 1.98 (32)
12.69 0.0047 2.70 (44)
3.13 0.0023 1.36 (22)
1.47 0.0016 0.90 (14)
1.73 0.0017 1.00 (16)
11.79 0.0045 2.62 (43)
7.45 0.0036 2.07 (34)
12.69 0.0047 2.70 (44)
Sodium alginate–CaCl2 (B-4)
Chitosan alginate–CaCl2 (B-5)
Multilayered beads (B-6)
The enzyme activity, protein content and specific activity for unimmobilized culture were 64.5 (EU), 0.01057 (g), and U (100%) respectively. The values within the parentheses indicate the % immobilization. All the errors were within 5% of SD.
A. Sharma et al. / Enzyme and Microbial Technology 48 (2011) 416–426
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Table 2 Comparison of enzyme activity (EU/g of beads), protein content (g/g beads), specific activity (U/g) and immobilization potential (%) of purified M. lylae carbonic anhydrase. Immobilization period
Immobilization matrix Chitosan–NaOH (B-1)
2h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 4h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 6h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 8h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 10 h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g)
Chitosan–KOH (B-2)
Chitosan–NH4 OH (B-3)
Sodium alginate–CaCl2 (B-4) 3.32 0.0005 6.65 (10)
Chitosan alginate–CaCl2 (B-5)
Multilayered beads (B-6)
10.8 0.0009 12.0 (18)
9.90 0.0009 11.0 (17)
41.94 0.0018 23.3 (35)
53.2 0.002 26.6 (40)
49.4 0.0019 26.0 (39)
61.6 0.0022 28.0 (43)
70.38 0.0023 30.6 (46)
66.0 0.0022 30.0 (45)
25.2 0.0014 18.0 (27)
28.95 0.0015 19.3 (29)
25.2 0.0014 18.0 (27)
153.68 0.0034 45.2 (68)
169.75 0.0035 48.5 (69)
169.46 0.0037 45.8 (73)
30 0.0015 20.0 (30)
41.9 0.0018 23.3 (35)
41.4 0.0018 23.0 (35)
165.2 0.0035 47.2 (71)
231.84 0.0042 55.2 (83)
189.24 0.0038 49.8 (75)
37.4 0.0017 22.0 (33)
62.92 0.0022 28.6 (43)
52.0 0.002 26.0 (39)
165.2 0.0035 47.2 (71)
231.84 0.0042 55.2 (83)
189.24 0.0038 49.8 (75)
29.92 0.0015 19.95 (30)
58.65 0.0021 27.93 (42)
52.0 0.002 26 (39)
The enzyme activity, protein content and specific activity for free purified enzyme were 305.5 (EU), 0.005 (g) and 66.5 U (100%), respectively. The values within the parentheses indicate the % immobilization. All the errors were within 5% of SD.
Table 2a Comparison of enzyme activity (EU/g of beads), protein content (g/g beads), specific activity (U/g) and immobilization potential (%) of whole cell M. lylae (carbonic anhydrase). Immobilization period
Immobilization matrix Chitosan–NaOH (B-1)
2h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 4h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 6h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 8h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 10 h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g)
Chitosan–KOH (B-2)
Chitosan–NH4 OH (B-3)
Sodium alginate–CaCl2 (B-4)
Chitosan alginate–CaCl2 (B-5)
Multilayered beads (B-6)
10.48 0.0045 2.33 (46)
6.84 0.0036 1.90 (37)
12.49 0.0049 2.53 (50)
1.28 0.0016 0.80 (16)
1.60 0.0017 0.89 (18)
2.00 0.002 1.00 (20)
13.26 0.0051 2.60 (52)
7.8 0.0039 2.00 (39)
16.24 0.0056 2.90 (57)
1.98 0.002 0.99 (20)
2.35 0.0022 1.07 (22)
2.73 0.0023 1.19 (23)
19.8 0.0062 3.20 (63)
9.28 0.0042 2.21 (43)
19.33 0.0061 3.17 (62)
2.90 0.0024 1.21 (24)
2.46 0.0022 1.12 (22)
3.43 0.0026 1.32 (26)
21.12 0.0064 3.30 (65)
12.54 0.0049 2.56 (50)
27.37 0.0073 3.75 (74)
4.38 0.0029 1.51 (30)
5.15 0.0032 1.61 (32)
6.55 0.0036 1.82 (36)
21.22 0.0064 3.30 (65)
13.0 0.0050 2.60 (51)
28.12 0.0074 3.80 (75)
4.80 0.0031 1.55 (31)
5.50 0.0032 1.72 (34)
6.20 0.0038 1.63 (38)
The enzyme activity, protein content and specific activity for unimmobilized culture were 50.1 (EU), 0.00988 (g) and 5.07 U (100%) respectively. The values within the parentheses indicate the % immobilization. All the errors were within 5% of SD.
imum immobilization was observed on B-4 (Table 1a, Table 2a, and Table 3a). The immobilization potential (%), enzyme activity (EU/g beads) and mass transfer (g/g beads) were determined for free cells (Table 1a, Table 2a and Table 3a). Maximum mass transfer (mg/mg beads) and enzyme activity (EU/g beads) were observed on chitosan–NH4 OH (B-3) beads for M. luteus 2 cell (0.0067 g/g beads; 26.46 EU/g beads), followed by M. lylae (0.0074 mg/mg beads; 27.12 EU/beads) cell and on multi-layered beads (B-6) for P. fragi cell (0.0047 g/g beads; 12.69 EU/g beads). Since only a maximum of 75% cell immobilization (M. lylae on B-3) was obtained compared to 89% enzyme immobilization of CA from P. fragi (B-2), immobilized cells were not considered for further study.
3.2. Effect of pH and temperature on stability of immobilized CA The stability of free and immobilized CA at different pH and temperature is illustrated in Table 4. The results show that at pH 8.0, 100% activity was retained for immobilized P. fragi CA, while 72% and 83% residual activity was retained for M. lylae and M. luteus 2 immobilized CA respectively. The immobilized CA from P. fragi showed 80% stability in the pH range 7.0–9.0, while M. luteus 2 and M. lylae immobilized CA showed 80% stability between the pH range 8.0–9.0 and 7.0–7.5, respectively. The temperature stability profile for immobilized CA from P. fragi showed above 80% residual activity between the temperature
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Table 3 Comparison of enzyme activity (EU/g of beads), protein content (g/g beads), specific activity (U/g) and immobilization potential (%) of purified M. luteus 2 carbonic anhydrase. Immobilization period
Immobilization matrix Chitosan–NaOH (B-1)
2h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 4h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 6h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 8h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 10 h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g)
Chitosan–KOH (B-2)
Chitosan–NH4 OH (B-3)
Sodium alginate–CaCl2 (B-4)
Chitosan alginate–CaCl2 (B-5)
Multilayered beads (B-6)
38.43 0.0018 21.35 (35)
48.2 0.0020 24.1 (40)
44.08 0.0019 23.2 (39)
11.7 0.001 11.7 (20)
11.60 0.001 11.6 (19)
10.8 0.0009 12.0 (18)
53.76 0.0021 25.6 (42)
87.90 0.0030 29.3 (59)
74.25 0.0027 27.5 (54)
22.20 0.0014 15.86 (27)
25.65 0.0015 17.1 (29)
25.2 0.0014 18.0 (27)
153.72 0.0036 42.7 (71)
176.28 0.0039 45.2 (78)
130.68 0.0033 39.6 (65)
33.18 0.0016 20.74 (32)
44.08 0.0019 23.2 (39)
45 0.0018 25.0 (36)
182.45 0.0041 44.5 (81)
215.04 0.0042 51.1 (84)
173.66 0.0038 45.7 (75)
44.27 0.0019 23.3 (38)
45.20 0.002 22.6 (40)
52.0 0.002 26.0 (39)
182.45 0.0041 44.5 (81)
215.04 0.0042 51.1 (84)
173.66 0.0038 45.7 (75)
44.27 0.0019 23.2 (38)
45.20 0.002 22.6 (40)
52.0 0.002 26 (39)
The enzyme activity, protein content and specific activity for free purified enzyme were 307.7 (EU), 0.005 (g) and U (100%), respectively. The values within the parentheses indicate the % immobilization. All the errors were within 5% of SD.
Table 3a Comparison of enzyme activity (EU/g of beads), protein content (g/g beads), specific activity (U/g) and immobilization potential (%) of whole cell M. luteus 2 (carbonic anhydrase). Immobilization period
Immobilization matrix Chitosan–NaOH (B-1)
2h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 4h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 6h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 8h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 10 h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g)
Chitosan–KOH (B-2)
Chitosan–NH4 OH (B-3)
Sodium alginate–CaCl2 (B-4)
Chitosan alginate–CaCl2 (B-5)
Multilayered beads (B-6)
9.93 0.0043 2.31 (41)
5.54 0.0031 1.79 (32)
12.42 0.0046 2.70 (48)
1.38 0.0015 0.92 (16)
1.42 0.0015 0.95 (16)
2.00 0.0018 1.10 (19)
11.33 0.0044 2.58 (46)
5.95 0.0032 1.86 (33)
14.75 0.0050 2.95 (52)
1.70 0.0017 1.00 (18)
1.96 0.0018 1.09 (19)
3.10 0.0023 1.35 (24)
17.98 0.0055 3.27 (57.9)
6.80 0.0034 2.00 (35.4)
17.93 0.0055 3.26 (57.8)
2.18 0.0019 1.15 (20.4)
2.38 0.0020 1.19 (21.1)
3.65 0.0025 1.46 (25.8)
17.98 0.0055 3.27 (57.9)
8.06 0.0037 2.18 (38.6)
25.74 0.0066 3.90 (69.1)
5.64 0.0031 1.82 (32.2)
5.37 0.0030 1.79 (31.7)
6.14 0.0032 1.92 (34)
18.76 0.0056 3.33 (59)
8.06 0.0037 2.18 (38.6)
26.46 0.0067 3.95 (70)
5.95 0.0032 1.86 (33)
6.04 0.0032 1.89 (33.5)
7.31 0.0035 2.09 (37)
The enzyme activity, protein content and specific activity for unimmobilized culture were 53.9 (EU), 0.00955 (g) and 5.64 U (100%) respectively. The values within the parentheses indicate the % immobilization. All the errors were within 5% of SD.
Table 4 Effect of pH and temperature on stability of immobilized CA on Chitosan–KOH beads. Immobilized CA (3 h)
P. fragi M. lylae M. luteus 2
Temperature (◦ C) (residual activity %)
pH (residual activity %) 7.0
7.5
87 (86) 89 (87) 69 (69)
93 (92) 88 (88) 78 (78)
8.0 100 (99) 72 (80) 83 (83)
8.5 90 (85) 68 (68) 91 (90)
9.0 84 (78) 60 (57) 89 (88)
35
40
87 (80) 81 (75) 94 (90)
84 (77) 79 (70) 92 (86)
45 80 (72) 76 (67) 91 (85)
50
55
74 (66) 70 (60) 87 (80)
68 (59) 64 (54) 84 (76)
The values within the parentheses indicate the residual activity of free enzyme (Sharma and Bhattacharya [13]). All the errors were within 5% of SD.
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421
Table 5 CO2 sequestration efficiency of CA immobilized on chitosan–KOH beads and evaluation of reusability potential in presence of CO2 saturated ASW+ SOx + NOx. The Ca2+ ion concentration was determined after precipitation (5 min). 35 ◦ C 0.2 mM
Control (without enzyme)
45 ◦ C 0.4 mM
Free enzyme
Single cycle
P. fragi CA (% efficiency for calcium utilization & mM of calcium ions utilized)
M. lylae CA (% efficiency for calcium utilization & mM of calcium ions utilized)
M. luteus 2 CA (% efficiency for calcium utilization & mM of calcium ions utilized)
35 ◦ C
45 ◦ C
35 ◦ C
45 ◦ C
35 ◦ C
45 ◦ C
38 [3.8]
53 [5.3]
34.5 [3.45]
48.8 [4.1]
31 [3.1]
44 [4.0]
Reusability cycles
01 02 03 04 05 06 07 08 09 10 Total mM of calcium ion utilized
Immobilized enzyme P. fragi CA (% efficiency for calcium utilization & mM of calcium ions utilized)
M. lylae CA (% efficiency for calcium utilization & mM of calcium ions utilized)
M. luteus 2 CA (% efficiency for calcium utilization & mM of calcium ions utilized)
35 ◦ C
45 ◦ C
35 ◦ C
45 ◦ C
35 ◦ C
45 ◦ C
25 [2.5] 25 [2.5] 18 [1.8] 15 [1.5] 10 [1.0] 07 [0.7] 03 [0.3] – – –
33 [3.3] 33 [3.3] 20 [2.0] 17 [1.7] 13 [1.3] 09 [0.9] 05 [0.5] – – –
19.5 [1.95] 19.5 [1.95] 14 [1.4] 12 [1.2] 08 [0.8] 05 [0.5] – – – –
26 [2.6] 26 [2.6] 17 [1.7] 15 [1.5] 10 [1.0] 07 [0.7] – – – –
17 [1.7] 17 [1.7] 12 [1.2] 10 [1.0] 06 [0.6] 02 [0.2] – – – –
23 [2.3] 23 [2.3] 16 [1.6] 14 [1.4] 09 [0.9] 05 [0.5] – – – –
10.3
13
1.8
10.1
6.4
8.0
Initial calcium concentration for each cycle was 10 mM. The values within the parentheses indicate concentration of calcium ions utilized. All the errors were within 5% SD.
range 35–45 ◦ C, while from M. luteus 2, 80% stability was recorded between 35 and 55 ◦ C. The immobilized M. lylae CA retained above 70% residual activity in the temperature range 35–50 ◦ C.
0.0667), M. lylae (0.0354; 0.0473) and M. luteus 2 (0.0302; 0.0406). During reusability 100% sequestration efficiency was retained for 2 cycles followed by gradual loss in sequestration potential.
3.3. Storage stability
3.5. FTIR analysis
The specific activities on day 1 for free CA from P. fragi, M. lylae and M. luteus 2 were determined as 70.6 U, 66.5 U and 61.0 U respectively. However, the specific activities after day 30 for free CA from P. fragi, M. lylae and M. luteus 2 were determined as 28.9 U, 27.5 U and 22.1 U, respectively. In contrast, the specific activities on day 1 for immobilized CA from P. fragi, M. lylae and M. luteus 2 were determined to be 62.8 U/g beads, 55.2 U/g beads and 51.42 U/g beads, respectively. Similarly, the specific activities on day 30 for immobilized CA from P. fragi, M. lylae and M. luteus 2 were determined to be 52.1 U/g beads, 45.8 U/g beads and 42.4 U/g beads respectively. The storage stability experiments showed that only 41% of residual activity is retained by free CA compared to 83% by immobilized CA.
The FTIR spectra indicated different peaks for chitosan at 3469 cm−1 , 3279 cm−1 , 3192 cm−1 , 3086 cm−1 , 2881 cm−1 , 2870 cm−1 , 1663 cm−1 and 1421 cm−1 (Fig. 1A). Similarly for chitosan–KOH, different peaks were obtained at 3679 cm−1 , 3261 cm−1 , 3190 cm−1 , 2924 cm−1 , 2862 cm−1 , 1678 cm−1 , and 1500 cm−1 (Fig. 1B). The FTIR spectra for free enzyme showed major peaks at 3284 cm−1 , 3072 cm−1 , 2980 cm−1 , 2929 cm−1 , 2879 cm−1 , 1674 cm−1 , 1647 cm−1 , 1527 cm−1 , 1521 cm−1 , and 1460 cm−1 (Fig. 2A) Correspondingly, the spectra for enzyme immobilized chitosan–KOH beads showed major peaks at 3749 cm−1 , 3421 cm−1 , 3275 cm−1 , 2949 cm−1 , 2858 cm−1 , 1683 cm−1 , 1523 cm−1 , 1521 cm−1 , and 1460 cm−1 (Fig. 2B).
3.4. CO2 sequestration efficiency and reusability potential
3.6. XRD analysis
Table 5 shows the percentage efficiency of calcium ion utilization as a measure of CO2 sequestration using immobilized CA from all the three strains. The immobilized CA from P. fragi showed sequestration potential up to 7 recycles, while immobilized CA from M. lylae and M. luteus 2 were effective for 6 recycles only. The total concentration (mM) of calcium ion utilization by 1 g of chitosan–KOH immobilized CA at 35 ◦ C and 45 ◦ C was determined for P. fragi (10.3 mM; 13 mM), M. lylae (7.8 mM; 10.1 mM), and M. luteus 2 (6.4 mM; 9.0 mM). Table 6 shows the total amount of CaCO3 (g) formed following reusability of immobilized CA at 35 ◦ C and 45 ◦ C for P. fragi (0.111; 0.152); M. lylae (0.081; 0.108) and M. luteus 2 (0.069; 0.093). Similarly the gram equivalent of CO2 present in CaCO3 at 35 ◦ C and 45 ◦ C was also calculated for P. fragi (0.0485;
Fig. 3 illustrates the X-ray diffraction pattern obtained for biomimetically produced CaCO3 . The major peaks at 2 = 29.4◦ and 27.08◦ were obtained. The other high intensity peaks were recorded at 2 = 27.1◦ , 32.7◦ , 24.9◦ , 43.8◦ . 3.7. SEM analysis The scanning electron micrographs of calcium carbonate formed by the enzyme are shown in Fig. 4. The image displayed hexagonal shaped crystals. Most of the crystals were of faceted rhombohedral shaped (calcite), however some crystals with spherical characteristics (vaterite) were also visible.
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Table 6 Conversion of CO2 (gram equivalent) into calcium carbonate using CA immobilized on chitosan–KOH beads in presence of CO2 saturated ASW + SOX + NOX. The total amount of calcium carbonate formed was determined after precipitation (5 min). 35 ◦ C 0.2 mM
Control (without enzyme)
45 ◦ C 0.4 mM
Free enzyme P. fragi CA
Single cycle
M. lylae CA
M. luteus 2 CA
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
0.042
0.0184
0.051
0.024
0.032
0.0140
0.041
0.0180
0.029
0.012
0.030
0.0167
Reusability cycles
Immobilized enzyme P. fragi CA
M. lylae CA
M. luteus 2 CA
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
01 02 03 04 05 06 07
0.027 0.027 0.018 0.016 0.012 0.008 0.003
0.0118 0.0118 0.0079 0.0070 0.0052 0.0035 0.0013
0.035 0.035 0.027 0.021 0.018 0.010 0.006
0.0154 0.0154 0.0118 0.0092 0.0079 0.0044 0.0026
0.020 0.020 0.016 0.012 0.009 0.004 –
0.0088 0.0088 0.0070 0.0052 0.0039 0.0017 –
0.029 0.029 0.018 0.014 0.010 0.006 –
0.0127 0.0127 0.0079 0.0079 0.0044 0.0026 –
0.018 0.018 0.013 0.010 0.007 0.003 –
0.0079 0.0079 0.0057 0.0044 0.0030 0.0013 –
0.024 0.024 0.017 0.014 0.009 0.005 –
0.0105 0.0105 0.0074 0.0061 0.0039 0.0022 –
Total amount of calcium carbonate formed and gram equivalent of CO2 present
0.111
0.0485
0.152
0.0667
0.081
0.0354
0.106
0.0482
0.069
0.0302
0.093
0.0406
All the errors were within 5% SD.
4. Discussion In the present study the maximum specific activity and immobilization potential for immobilized CA from P. fragi (62.8 U/g beads; 89%), M. lylae (55.2 U/g beads; 83%) and M. luteus 2 (51.2 U/g beads; 84%) was obtained on chitosan–KOH. The chitosan derived beads; showed higher immobilization efficiency compared to alginate derived bead and multilayered beads for CA from all the three isolates. Chitosan has hydrophilic, hydroxyl and amino groups that probably facilitate the adsorption of enzyme on the matrix, whereas in alginate or multilayered beads, these groups are either absent or masked leading to less adsorption of enzyme on the matrix and correspondingly lower immobilization efficiency. The findings of Prabhu et al. [15] also substantiated our results. Successful cell immobilization was achieved on all the six materials. Maximum specific activity and efficiency for P. fragi (2.7 U/g beads; 44%) was obtained on multilayered beads. Contrastingly maximum specific activity and efficiency was obtained for M. lylae (3.76 U/g beads; 74%) and M. luteus 2 (3.95 U/g beads; 70%) on chitosan–NH4 OH. The differential adsorption of Gram negative P. fragi and Gram positive M. lylae and M. luteus on chitosan derived beads (chitosan–NaOH, chitosan–KOH and chitosan–NH4 OH) and alginate derived beads is of significant importance. Chitosan is a heteropolymer of glucosamine and acetyl glucosamine units and has many hydrophilic groups. The cell wall of Gram positive bacteria consists mostly of peptidoglycan which is a polymer of N-acetyl glucosamine and N-acetyl muramic acid, amino acids and techoic acid along with surface exposed carbohydrates [21]. Thus, it could be rationally suggested that presence of hydrophilic groups on surface of Gram positive bacteria and chitosan derived beads (chitosan–NaOH, chitosan–KOH and chitosan–NH4 OH) facilitated the bacterial adsorption on these matrix. However, sodium alginate–CaCl2 , chitosan–alginate–CaCl2 and multilayered beads have a more hydrophobic character associated with them thereby non-facilitating the binding of Gram positive bacteria. The higher immobilization efficiency of P. fragi on multilayered beads and alginate beads can be justified from the fact that Gram nega-
tive bacteria have an outer membrane consisting of phospholipids and lipopolysaccharide which imparts a hydrophobic character to these bacteria [22]. The adsorption is thus facilitated by hydrophobic interaction between P. fragi multilayered and alginate derived beads. The study thus substantiated the choice of proper matrix for immobilization of cells and enzymes and corroborated the importance of designing and synthesis of different materials based on the type of sample to be immobilized. The pH profile for immobilized CA from P. fragi, M. lylae and M. luteus 2 was similar to that of the free enzyme. However, there was slight increase in stability of all the three immobilized CA at pH 8.0–9.0. The temperature stability of the immobilized CA showed marked improvement compared to free CA. A maximum increase of 1.08–1.18 fold stability between 35–55 ◦ C for immobilized CA was observed for M. lylae followed by P. fragi (1.08–1.15) and M. luteus 2 (1.04–1.10) compared to free CA. The operational stability of the immobilized CA at elevated temperature has high degree of functional and economical significance associated with it. Bond et al. [2] had already reported the optimum pH range of 8.0–9.0 and temperature range of 35–45 ◦ C as vital components for successful biomimetic sequestration process. The storage stability of immobilized CA in aqueous medium was 2.02 fold higher compared to free CA. This acts as added incentive for the bulk production and storage of immobilized enzyme. The peaks at 3469 cm−1 and 3679 cm−1 for chitosan flakes and chitosan–KOH indicated the stretching vibrations, while the peaks at 3279 cm−1 and 3281 cm−1 for chitosan flakes and chitosan–KOH indicated the O–H stretching vibrations. The N–H and O–H bonds play an important role in hydrophilic interactions like hydrogen bonding and Vander walls interaction, similarly the FTIR spectrum of free enzyme represents the N–H, O–H and C–H stretching vibrations at 3700 cm−1 , 3284 cm−1 and 3072 cm−1 respectively that could be involved in the hydrophilic interactions. The FTIR spectra of CA immobilized on chitosan–KOH also showed the presence of peaks at 3479 cm−1 and 3421 cm−1 corresponding to stretching vibrations due to N–H and O–H groups respectively. The C O stretching at 2924 cm−1 and 2862 cm−1 in chitosan flakes
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Fig. 1. FTIR spectra of (A) chitosan flakes and (B) chitosan–KOH beads.
and chitosan KOH beads was deemed important for hydrogen bonding as they were also identified in free enzyme (2960 cm−1 , 2929 cm−1 and 2379 cm−1 ) and immobilized CA beads (2924 cm−1 and 2858 cm−1 ). The distinct peaks in free enzyme at 1527 cm−1 and 1521 cm−1 correspond to N–H bending vibrations due to presence of amides and/or secondary amines; while peak at 1460 cm−1 indicated the presence of aromatic ring with low degree of substitution (could be attributed to the presence of aromatic amino acids in the enzyme). The FTIR spectra of immobilized enzyme are characterized by N–H bending peaks at 1523 cm−1 and 1512 cm−1 and aromatic ring substitution at 1460 cm−1 thus establishing the successful immobilization of carbonic anhydrase on chitosan–KOH beads. The presence of O–H and N–H groups both on enzyme and immobilized beads confirmed the adsorption due to hydrophilic interactions. At 35 ◦ C, 70.6 U of free P. fragi CA, 66.5 U of free M. lylae CA, and 61.0 U of free M. luteus 2 CA effectively sequestered, 3.8 mM, 3.45 mM, and 3.11 mM respectively, of calcium ions into calcium carbonate. However, using the same U/g immobilized CA from P. fragi, M. lylae, and M. luteus 2 on chitosan–KOH beads sequestered, 10.3 mM, 7.8 mM and 6.4 mM respectively, of calcium ions into calcium carbonate. The reusability of immobilized enzyme allows its repeated use compared to the free enzyme which is lost after single cycle. Thus for any concentration of immobilized CA corresponding
to same concentration of free CA, the amount of CO2 sequestered will always be high. In the present study, the amount of CaCO3 formed and gram equivalent of CO2 present in CaCO3 was 2 fold higher by immobilized CA compared to free CA from all the three strains. Similar phenomenon was also observed with the utilization of calcium ions. The highest sequestration efficiency (∼2.7 fold) was achieved with immobilized CA from P. fragi. Increase in sequestration efficiency at 45 ◦ C was evident for both free and immobilized enzyme. The fact that modest heating (∼50 ◦ C) overcomes inhibition of precipitation [4] in artificial sea water has been proved beyond doubt [13]. These results are very encouraging and advocate the implementation of immobilized enzyme systems at on-site scrubber. A constant loss in sequestration efficiency was observed during reusability of immobilized enzyme. This phenomenon is attributable to the inhibition of enzyme activity due to the presence of different anions in ASW and/or gradual loss of enzyme from the matrix, the latter however needs experimental explication. In an on-site scrubber SOx and NOx are considered to be major inhibitory ions. During our previous study [13], we found that free CA from P. fragi, M. lylae, and M. luteus 2 retained 83%, 92%, and 60% respectively of their residual activity at 0.1 M concentration of sulphate ions, however considerable inhibition was observed for free CA from P. fragi (30%), M. lylae (27%) and M. luteus
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Fig. 2. FTIR spectra of (A) enzyme (carbonic anhydrase) and (B) CA immobilized on chitosan–KOH beads.
(56%) at 0.1 M concentration of nitrate ions. Also Cl− (200 mM), Br− (50 mM), I− (50 mM), and HCO3 − (50 mM), were found to have significant inhibitory effect on all the three CAs. The inhibition profile of each ion varied with CA from three isolates [13]. The calcite phase was found to be the major form of CaCO3 obtained from biomimetic CO2 sequestration using indigenous immobilized CA. The diffraction peaks at 2 = 29.4◦ and 43.8◦ cor-
Fig. 3. XRD spectra of biomimetically produced CaCO3 .
responded to the calcite phase. The other major peaks at 27.08◦ , 27.14◦ and 32.7◦ corresponded to the vaterite phase of CaCO3 . Similar results have been reported by Favre et al. [20] and Li et al. [23]. The SEM images confirmed the presence of both vaterite and calcite phase. A hexagonal structure is obtained with both the phases [24,25], however complex packing in vaterite is illustrated with the spherical appearance of the crystal. The calcite crystals displayed well defined faceted rhombohedral characteristics. In the present study calcite was found to be the most dominant form as it is thermodynamically more stable compared to metastable vaterite at pH < 10 (sequestration was carried out at pH 9.5), however vaterite is dominant above pH 10 [20]. Both Favre et al. [20] and Li et al. [23] have shown that calcite phase is dominant when the precipitation occurs in presence of CA. Phase transformation in crystallographic structure of solid particles with time is a phenomena commonly associated with solid particles grown in liquid medium [26]. Three different mechanisms advocate such solid transformation. The first mechanism involves change from nonuclear mode to either polynuclear mode or mononuclear mode, which was not observed in the present study. The second mechanism of ageing involves dissolution of first burst particle followed by reprecipitation to a more stable calcite phase with time. The third mechanism combines ageing with hydrolysis of products in presence of side chains of proteins [26,27]. Since the protein concentration is very low and the precipitation
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Fig. 4. (A–D) SEM images of biomimetically produced CaCO3 , showing both the calcite and vaterite phases using P. fragi CA immobilized on chitosan–KOH beads.
reaction was initiated in chamber without enzyme, this mechanism seems unlikely. The phase transition is evident in Fig. 4B which clearly shows the highly porous nature of vaterite particles, while the thin planar non-porous crystals of calcite are visible, surfacing from the vaterite particles, thus supporting the dissolution reprecipitation mechanism of ageing. CA accelerates the formation of bicarbonate ions by lowering the activation energy required for hydration of CO2 . The conversion of CO2 into H2 CO3 is the rate controlling step catalyzed by CA, while the formation of HCO3 − is nearly diffusion controlled [6]. However, the conversion of HCO3 − to CO3 2− is pH dependent [4,20]. An enzyme can only increase the rate of the reaction, but could not alter it, thus CA is deemed to accelerate the formation of bicarbonate ions. Under the precipitation conditions (pH 9.0–9.5) bicarbonate ions exist in equilibrium with carbonate ions and in presence of calcium ions result in calcium carbonate precipitation. Thus CA plays the important role in hydration reaction corresponding to calcium carbonate formation, calcite being the dominant form [20]. Thus application of an effective immobilization system operating at low mass (protein) values under process parameters will open up a new avenue for cost effective sequestration of CO2 into CaCO3 in an onsite scrubber.
5. Conclusions The study highlighted the effectiveness of different immobilization matrices corresponding to both Gram positive and Gram negative bacteria. The study for the first time reports the biomimetic sequestration of CaCO3 using immobilized CA from three indigenous strains under conditions simulating an on-site scrubber. The CO2 sequestration potential and reusability of immobilized CA compared to free CA provides this system an immense advantage and towering edge along with economic relevance and commercial utility.
Acknowledgements The authors are grateful to DBT, New Delhi for providing the financial assistance. A. Bhattacharya is thankful to Council of Scientific and Industrial Research (CSIR), New Delhi for providing the CSIR-SRF fellowship. The FTIR facility provided by Dr. Anjali Bajpai, Department of Chemistry, Govt. Model Science College, Jabalpur (M.P.) and XRD and SEM facility by Dr. Sadhna Rayalu, Head EMD, National Environmental Engineering Research Institute (NEERI), Nagpur is highly acknowledged. The authors are also thankful to the Head, Department of Biological Science, Rani Durgavati University, Jabalpur (M.P.), for providing laboratory facilities.
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Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Biomimetic CO2 sequestration using purified carbonic anhydrase from indigenous bacterial strains immobilized on biopolymeric materials Anjana Sharma ∗ , Abhishek Bhattacharya, Ankita Shrivastava Bacteriology Laboratory, Department of P.G. Studies and Research in Biological Science, Rani Durgavati University, Pachpedi, Jabalpur 482001, Madhya Pradesh, India
a r t i c l e
i n f o
Article history: Received 3 October 2010 Received in revised form 2 February 2011 Accepted 2 February 2011 Keywords: Biomimetic CO2 -sequestration Immobilization Carbonic anhydrase P. fragi Indigenous
a b s t r a c t The present study deals with immobilization of purified CA and whole cell of Pseudomonas fragi, Micrococcus lylae, and Micrococcus luteus 2 on different biopolymer matrices. Highest enzyme immobilization was achieved with P. fragi CA (89%) on chitosan–KOH beads, while maximum cell immobilization was achieved with M. lylae (75%) on chitosan–NH4 OH beads. A maximum increase of 1.08–1.18 fold stability between 35 and 55 ◦ C was observed for M. lylae immobilized CA. The storage stability was improved by 2.02 folds after immobilization. FTIR spectra confirmed the adsorption of CA on chitosan–KOH beads following hydrophilic interactions. Calcium carbonate precipitation was achieved using chitosan–KOH immobilized P. fragi CA. More than 2 fold increase in sequestration potential was observed for immobilized system as compared to free enzyme. XRD spectra revealed calcite as the dominant phase in biomimetically produced calcium carbonate. © 2011 Elsevier Inc. All rights reserved.
1. Introduction The rising concentration of green house gases (GHGs), and in particular carbon dioxide (CO2 ) due to anthropogenic interventions has led to several undesirable consequences such as global warming and related changes. The most easily addressable source of CO2 is that from the generation of electric power, and particularly that from the coal burning stations, since they comprise a relatively small number of very large stationary sources [1]. Coal fired power plants currently account for little more than half the electricity generation and it is unlikely that there will be a dramatic change in this situation in the next twenty years [2,3]. Conversion of CO2 into solid carbonates offers the possibility of a safe and stable ecofriendly product for long term carbon sequestration [2]. Precipitation from aqueous solution occurs at a suitable supersaturation of calcium and carbonate ions [4]. The hydration of CO2 to form carbonic acid is the rate-limiting step in the conversion of CO2 into carbonate ions, which has a forward reaction constant of 6.2 × 10−3 s at 25 ◦ C [5,6]. This hydration reaction is catalyzed by carbonic anhydrase (CA) at or near diffusion controlled limit [7]. Carbonic anhydrase (CA) is one of the fastest enzymes that catalyses CO2 -hydration reaction with typical rates between 104 and 106 reactions per second for different forms of this enzyme [8]. It is a zinc metalloenzyme reported to be present in animals, plants and
microorganisms [9–12]. The successful precipitation of CO2 (aq) into CaCO3 in the presence of calcium ions through biomimetic approach involving, indigenous carbonic anhydrase [13] and commercial Bovine Carbonic anhydrase (BCA) [14] has been proved in principle. However, commercial application of this approach warrants the immobilization of CA on to a suitable immobilization matrix for application at an onsite scrubber [14]. The process of immobilization confines enzymes/whole cells to a phase distinct from the one in which the substrates and the products are present [15]. Chitosan and sodium alginate are inert materials that have been used for immobilization of enzymes and microorganism [15]. Immobilization enhances the efficiency of the process by allowing reuse of enzyme coupled with easy removal of the products from the reaction mixture. The physical methods especially adsorption, have an advantage over the chemical methods for immobilization of enzymes onto carriers in that it is simple, less expensive and can retain high catalytic activity [16]. CO2 sequestration using immobilized BCA and indigenous CA under different process parameters is under research phase [13,15] and an objectively defined study involving indigenous bacterial CA immobilized on inert matrix is the need of the hour. The present study thus aims at immobilization of both CA and bacterial cells of indigenous origin on chitosan and alginate based materials and to assess their CO2 sequestration potential. 2. Materials and methods
∗ Corresponding author. Tel.: +91 761 2416667; fax: +91 761 2603752; mobile: +91 9425155323. E-mail address: [email protected] (A. Sharma). 0141-0229/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2011.02.001
Artificial sea water (ASW) and chitosan were purchased from Sigma–Aldrich, St Louis, MO, USA. Sodium alginate was purchased from Himedia Mumbai, India. All the buffers used in the study were purchased from Himedia Mumbai, India. The sodium
A. Sharma et al. / Enzyme and Microbial Technology 48 (2011) 416–426 and potassium salts of chloride, bromide, iodide, fluoride, carbonate, sulphate and nitrate were purchased from Sisco Research Laboratory, Mumbai, India.
2.1. Synthesis of materials Chitosan–NaOH beads (B-1), chitosan–KOH beads (B-2), chitosan–NH4 OH beads (B-3), sodium alginate–CaCl2 beads (B-4), chitosan–sodium alginate–CaCl2 beads (B-5) and multilayered beads (B-6), were prepared following the method of Prabhu et al. [15].
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2.5. Effect of pH and temperature on immobilized CA The stability of the CA immobilized on chitosan–KOH beads was assayed using two different buffer systems, 50 mM phosphate buffer (pH 7.0, 7.5 and 8.0) and Tris–HCl buffer (pH 8.5 and 9.0) at 37 ◦ C for 3 h. The temperature stability of immobilized CA on chitosan–KOH beads was determined by carrying out the enzyme reactions at different temperatures (35–55 ◦ C) and pH 8.0 for 3 h. The enzyme activity at the start of the experiment was taken as 100% and the residual activity was determined after incubation. 2.6. Storage stability
2.2. Source of the bacterial strain and carbonic anhydrase The indigenous purified CA from Pseudomonas fragi (BGCC = / 1077), Micrococcus lylae (BGCC = / 1078), and Micrococcus luteus 2 (BGCC = / 1079) were obtained from Bacterial Germplasm Collection Centre (BGCC), Bacteriology Laboratory, Department of P.G. Studies and Research in Biological Science, R.D. University, Jabalpur (M.P.), India.
2.3. Immobilization 2.3.1. Immobilization of enzyme Lyophilized CA (5 mg) was dissolved in 5 ml of 50 mM Tris–HCl (pH 8.0) and mixed separately with 1 g of the immobilization matrix. The experimental setup containing 3 sets was designed. Each set with five separate tubes contained enzyme immobilization matrix suspension for P. fragi CA, M. lylae CA and M. luteus 2 CA, separately. The suspension in all the three sets was agitated (120 rpm) at 4 ◦ C and the immobilization efficiency was determined at every 2 h interval up to 10th h, using a single tube containing the suspension each from the three sets designated for CA from the three indigenous strains. After immobilization the beads were separated from the suspension and enzyme activity (EU) and protein concentration (mg) in the supernatant was determined. The direct estimation of CO2 hydration activity on beads after immobilization was carried out using 200 mg immobilized enzyme. The total enzyme activity on the beads was also determined as the difference between the total enzyme activity of free enzyme in solution before immobilization and the total enzyme activity present in the solution after immobilization. The total enzyme activity of immobilized CA was expressed as EU/g beads and the mass transfer of enzyme on immobilized beads was determined as amount of protein retained on beads after immobilization i.e. difference in protein concentration before and after immobilization in the suspension and was expressed as g protein/g beads. The specific activity (U) [where, U = EU/mg protein], on the immobilized beads was also determined and was expressed as U/g beads. The immobilization potential (%) was determined in terms of specific activity retained on beads compared to that of free enzyme (100%).
2.3.2. Immobilization of microorganisms Culture (100 ml) of each strain (A600 nm = 1.2) and 1 g of different immobilization matrices was added into flask and incubated at 37 ◦ C for 10 h with 2 h interval at 120 rpm. After incubation the slurry was centrifuged and the beads were collected, washed thoroughly with sterile distilled water and suspended in Tris–HCl buffer (50 mM, pH 8.0). The sample was sonicated thrice for 10 s with 30 s intervals followed by centrifugation at 10,000 rpm for 10 min [15]. Similarly the culture (A600 nm = 1.2) without any immobilization matrix was centrifuged and suspended in Tris–HCl buffer (50 mM, pH 8.0), followed by sonication and centrifugation as described above. The supernatant obtained was used to determine the enzyme activity (EU) and protein concentration (mg). The total enzyme activity of immobilized cells was expressed as EU/g beads and the mass transfer of cells on immobilized beads was determined as amount of protein retained on beads after immobilization and was expressed as g protein/g beads. The specific activity (U) [where, U = EU/mg protein], on the immobilized beads was also determined and was expressed as U/g beads. The immobilization potential (%) was determined in terms of specific activity retained on beads compared to that of free culture (100%).
2.4. Determination of carbonic anhydrase activity The method of Sharma and Bhattacharya [13] was followed with certain modifications. In brief, carbon dioxide saturated water prepared by introducing CO2 (100 kPa) in 500 ml of Milli Q grade pure water for 1 h at 4 ◦ C. CO2 saturated water (3 ml) was immediately added to 2 ml of Tris–HCl buffer (100 mM; pH 8.3) [13], and 0.1 ml of free purified CA (1 mg/ml stock), 0.1 ml of supernatant left after the immobilized enzyme was separated from suspension and 0.1 ml of supernatant derived after sonication of cell culture and immobilized cells were transferred immediately but separately to determine the enzyme activity. The time required for the pH change 8.0–7.0 (t) was measured. The time required for the pH change (8.0–7.0) was used as control (tc ), when buffer was substituted for test sample. The enzyme assay was carried out at 4 ◦ C. The Wilbur Anderson units were calculated with the equation (tc − t)/t [17]. The protein content was determined by the method of Lowry et al. [18].
The storage stability of both the free enzyme and immobilized enzyme was determined by storing them for 30 days at 4 ◦ C. The residual activity was determined following the standard enzyme assay method. 2.7. Characterization of the immobilization matrix and immobilized enzyme FTIR spectra of chitosan flakes, chitosan–KOH beads, free carbonic anhydrase and CA immobilized on chitosan–KOH beads mixed with KBr pellets were recorded on Shimadzu FTIR spectrophotometer (V-110). Spectra of all the materials were scanned in the range of 400–4000 cm−1 . 2.8. Determining CO2 sequestration efficiency using immobilized carbonic anhydrase CO2 saturated solution using artificial sea water (ASW) was prepared at room temperature as described under Section 2.4. ASW was supplemented with K2 SO4 (0.1 M) and KNO3 (0.1 M) to study the effect of SOx and NOx on CA activity and sequestration efficiency. CO2 saturated solution (10 ml) was mixed with 1 ml of Tris buffer (pH 8.3) containing free CA (100 g) from all three bacterial strains separately for 15 min. The bicarbonate solution was released into another vessel through a valve containing 10 ml of CaCl2 solution (at a final concentration of 10.0 mM). To the above mixture, Tris-buffer pH 9.5 (2 ml, 1 M) was immediately added. The reaction mixture was incubated at 35 ◦ C and 45 ◦ C, respectively for 5 min to allow precipitation of CaCO3 and the amount of CaCO3 formed was determined [13]. The control experiment was carried out in the absence of enzyme and the results were expressed in terms of mg CaCO3 formed following control correction. CA from all the three indigenous isolates immobilized on chitosan–KOH beads was used for sequestration studies by replacing the free enzyme in the reaction chamber. The sequestration efficiency was evaluated by determining the ionic concentration of calcium present before and after carbonate precipitation following the method of Kolthof et al. [19]. The difference in calcium concentration was considered to be the amount of calcium utilized in formation of calcium carbonate. An enzyme free system was used as control for comparison. The percentage efficiency of calcium ion utilized was also calculated and results were reported following control correction. The weight of calcium carbonate was also calculated and gm equivalents of CO2 present in CaCO3 were also determined. 2.9. Reusability of immobilized CA for CO2 sequestration The immobilized CA was evaluated for reusability in 10 batch process. The reaction of CO2 saturated solution with immobilized enzyme was carried out in a chamber separate from the vessel used for CaCO3 precipitation. The CA beads were rinsed with distilled water to neutralize the pH and to remove any excess ions. The chamber is separated from the CO2 reservoir and precipitation vessel being connected through a two way valve allows its easy removal, washing of beads and refining into the main apparatus for further reuse. 2.10. XRD analysis The X-ray diffraction spectra of biomimetically produced CaCO3 were recorded on a Rigaku Miniflex II instrument using Cu K␣ radiation ( = 0.15406 nm) operated at 30 kV and 15 mA. 2.11. SEM analysis Scanning electron microscopy was performed on selected carbonate samples generated biomimetically. A JEOL JSM 5800 LV electron microscope under an electrical tension of 20 kV was used for this purpose. A small solid piece of each sample to be examined was placed on a sample holder covered with a carbon tab and metalized with gold for 2.5 min in a cathodic atomizer blazer (20).
3. Results The specific activity of purified CA (1 mg/ml) from P. fragi (70.6 U), M. lylae (66.5 U) and M. luteus 2 (61.0 U) was determined following CO2 hydration assay.
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Table 1 Comparison of enzyme activity (EU/g of beads), protein content (g/g beads), specific activity (U/g) and immobilization potential (%) of purified P. fragi carbonic anhydrase. Immobilization period
Immobilization matrix Chitosan–NaOH (B-1)
2h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 4h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 6h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 8h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 10 h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g)
Chitosan–KOH (B-2)
Chitosan–NH4 OH (B-3)
Sodium alginate–CaCl2 (B-4)
Chitosan alginate–CaCl2 (B-5)
Multilayered beads (B-6)
46.98 0.0018 26.1 (37)
62.16 0.0021 29.6 (42)
56.4 0.002 28.2 (40)
12.06 0.0009 13.4 (19)
13.90 0.0001 13.9 (20)
11.7 0.0009 13 (18)
88.25 0.0025 35.3 (50)
120.64 0.0029 41.6 (59)
102.87 0.0027 38.1 (54)
24.83 0.0013 19.1 (27)
28.56 0.0014 20.4 (29)
28 0.0014 20 (28)
175.35 0.0035 50.1 (71)
214.89 0.0039 55.1 (78)
192.4 0.0037 52.0 (75)
31.2 0.0016 19.5 (32)
52.25 0.0019 27.5 (39)
33.0 0.0015 22 (31)
229.2 0.0004 57.3 (80)
276.32 0.0044 62.8 (89)
249.06 0.0042 59.3 (84)
50.92 0.0019 26.8 (38)
62.16 0.0021 29.6 (42)
60.9 0.0021 29 (41)
234.52 0.0041 57.2 (81)
276.32 0.0044 62.8 (89)
249.06 0.0042 59.3 (84)
50.92 0.0019 26.8 (38)
62.16 0.0021 29.6 (42)
42.5 0.0017 25 (35)
The enzyme activity, protein content and specific activity for free purified enzyme were 353 (EU), 0.005 (g) and 70.6 U (100%) respectively. The values within the parentheses indicate the % immobilization. All the errors were within 5% of SD.
3.1. Screening of materials for immobilization of enzyme and cells Determination of enzyme and cell immobilization in terms of specific activity showed that maximum CA immobilization was achieved after 8 h of incubation; while maximum cell immobilization was achieved following 10 h of incubation irrespective of the type of immobilization material used. Maximum immobilization of CA from all the three strains was obtained on B-2 followed by B-3 and B-1, less than 50% immobilization was achieved with B-4, B5 and B-6 materials (Table 1, Table 2, and Table 3). CA immobilized on chitosan–KOH (B-2) was used for further
studies. The immobilization potential (%), enzyme activity (EU/g beads) and mass transfer (g/g beads) were determined for CA from all the three strains (Table 1, Table 2, and Table 3). Maximum mass transfer (g/g beads) and enzyme activity (EU/g beads) was observed on chitosan–KOH (B-2) beads for CA from P. fragi (0.0044 g/g beads; 276.32 EU/g beads) followed by M. lylae (0.0042 g/g beads; 231.84 EU/g beads) and M. luteus 2 (0.0042 g/g beads; 215.04 EU/g beads). Maximum cell immobilization for P. fragi was observed on B-6, followed by B-4 and B-5. Minimum immobilization was observed on B-2. However, maximum cell immobilization for M. lylae and M. luteus 2 was observed on B-3 followed by B-1 and B-2. Min-
Table 1a Comparison of enzyme activity (EU/g of beads), protein content (g/g beads), specific activity (U/g) and immobilization potential (%) of whole cell P. fragi (carbonic anhydrase). Immobilization period
2h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 4h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 6h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 8h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 10 h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g)
Immobilization matrix Chitosan–NaOH (B-1)
Chitosan–KOH (B-2)
Chitosan–NH4 OH (B-3)
1.42 0.0016 0.89 (15)
0.19 0.0006 0.32 (5.3)
0.36 0.0008 0.45 (7.3)
3.17 0.0023 1.38 (22)
3.36 0.0024 1.40 (23)
3.40 0.0024 1.42 (23)
2.58 0.0021 1.23 (20)
0.40 0.0008 0.48 (7.8)
0.57 0.0010 0.57 (9.3)
3.70 0.0025 1.48 (24)
3.28 0.0024 1.37 (22)
4.41 0.0027 1.63 (26)
3.10 0.0023 1.35 (22)
1.04 0.0013 0.80 (13)
1.32 0.0015 0.88 (14)
9.28 0.0040 2.32 (38)
6.73 0.0034 1.98 (32)
9.76 0.0041 2.38 (39)
3.13 0.0023 1.36 (22)
1.35 0.0015 0.90 (14)
1.7 0.0017 1.00 (16)
11.79 0.0045 2.62 (43)
6.73 0.0034 1.98 (32)
12.69 0.0047 2.70 (44)
3.13 0.0023 1.36 (22)
1.47 0.0016 0.90 (14)
1.73 0.0017 1.00 (16)
11.79 0.0045 2.62 (43)
7.45 0.0036 2.07 (34)
12.69 0.0047 2.70 (44)
Sodium alginate–CaCl2 (B-4)
Chitosan alginate–CaCl2 (B-5)
Multilayered beads (B-6)
The enzyme activity, protein content and specific activity for unimmobilized culture were 64.5 (EU), 0.01057 (g), and U (100%) respectively. The values within the parentheses indicate the % immobilization. All the errors were within 5% of SD.
A. Sharma et al. / Enzyme and Microbial Technology 48 (2011) 416–426
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Table 2 Comparison of enzyme activity (EU/g of beads), protein content (g/g beads), specific activity (U/g) and immobilization potential (%) of purified M. lylae carbonic anhydrase. Immobilization period
Immobilization matrix Chitosan–NaOH (B-1)
2h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 4h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 6h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 8h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 10 h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g)
Chitosan–KOH (B-2)
Chitosan–NH4 OH (B-3)
Sodium alginate–CaCl2 (B-4) 3.32 0.0005 6.65 (10)
Chitosan alginate–CaCl2 (B-5)
Multilayered beads (B-6)
10.8 0.0009 12.0 (18)
9.90 0.0009 11.0 (17)
41.94 0.0018 23.3 (35)
53.2 0.002 26.6 (40)
49.4 0.0019 26.0 (39)
61.6 0.0022 28.0 (43)
70.38 0.0023 30.6 (46)
66.0 0.0022 30.0 (45)
25.2 0.0014 18.0 (27)
28.95 0.0015 19.3 (29)
25.2 0.0014 18.0 (27)
153.68 0.0034 45.2 (68)
169.75 0.0035 48.5 (69)
169.46 0.0037 45.8 (73)
30 0.0015 20.0 (30)
41.9 0.0018 23.3 (35)
41.4 0.0018 23.0 (35)
165.2 0.0035 47.2 (71)
231.84 0.0042 55.2 (83)
189.24 0.0038 49.8 (75)
37.4 0.0017 22.0 (33)
62.92 0.0022 28.6 (43)
52.0 0.002 26.0 (39)
165.2 0.0035 47.2 (71)
231.84 0.0042 55.2 (83)
189.24 0.0038 49.8 (75)
29.92 0.0015 19.95 (30)
58.65 0.0021 27.93 (42)
52.0 0.002 26 (39)
The enzyme activity, protein content and specific activity for free purified enzyme were 305.5 (EU), 0.005 (g) and 66.5 U (100%), respectively. The values within the parentheses indicate the % immobilization. All the errors were within 5% of SD.
Table 2a Comparison of enzyme activity (EU/g of beads), protein content (g/g beads), specific activity (U/g) and immobilization potential (%) of whole cell M. lylae (carbonic anhydrase). Immobilization period
Immobilization matrix Chitosan–NaOH (B-1)
2h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 4h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 6h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 8h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 10 h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g)
Chitosan–KOH (B-2)
Chitosan–NH4 OH (B-3)
Sodium alginate–CaCl2 (B-4)
Chitosan alginate–CaCl2 (B-5)
Multilayered beads (B-6)
10.48 0.0045 2.33 (46)
6.84 0.0036 1.90 (37)
12.49 0.0049 2.53 (50)
1.28 0.0016 0.80 (16)
1.60 0.0017 0.89 (18)
2.00 0.002 1.00 (20)
13.26 0.0051 2.60 (52)
7.8 0.0039 2.00 (39)
16.24 0.0056 2.90 (57)
1.98 0.002 0.99 (20)
2.35 0.0022 1.07 (22)
2.73 0.0023 1.19 (23)
19.8 0.0062 3.20 (63)
9.28 0.0042 2.21 (43)
19.33 0.0061 3.17 (62)
2.90 0.0024 1.21 (24)
2.46 0.0022 1.12 (22)
3.43 0.0026 1.32 (26)
21.12 0.0064 3.30 (65)
12.54 0.0049 2.56 (50)
27.37 0.0073 3.75 (74)
4.38 0.0029 1.51 (30)
5.15 0.0032 1.61 (32)
6.55 0.0036 1.82 (36)
21.22 0.0064 3.30 (65)
13.0 0.0050 2.60 (51)
28.12 0.0074 3.80 (75)
4.80 0.0031 1.55 (31)
5.50 0.0032 1.72 (34)
6.20 0.0038 1.63 (38)
The enzyme activity, protein content and specific activity for unimmobilized culture were 50.1 (EU), 0.00988 (g) and 5.07 U (100%) respectively. The values within the parentheses indicate the % immobilization. All the errors were within 5% of SD.
imum immobilization was observed on B-4 (Table 1a, Table 2a, and Table 3a). The immobilization potential (%), enzyme activity (EU/g beads) and mass transfer (g/g beads) were determined for free cells (Table 1a, Table 2a and Table 3a). Maximum mass transfer (mg/mg beads) and enzyme activity (EU/g beads) were observed on chitosan–NH4 OH (B-3) beads for M. luteus 2 cell (0.0067 g/g beads; 26.46 EU/g beads), followed by M. lylae (0.0074 mg/mg beads; 27.12 EU/beads) cell and on multi-layered beads (B-6) for P. fragi cell (0.0047 g/g beads; 12.69 EU/g beads). Since only a maximum of 75% cell immobilization (M. lylae on B-3) was obtained compared to 89% enzyme immobilization of CA from P. fragi (B-2), immobilized cells were not considered for further study.
3.2. Effect of pH and temperature on stability of immobilized CA The stability of free and immobilized CA at different pH and temperature is illustrated in Table 4. The results show that at pH 8.0, 100% activity was retained for immobilized P. fragi CA, while 72% and 83% residual activity was retained for M. lylae and M. luteus 2 immobilized CA respectively. The immobilized CA from P. fragi showed 80% stability in the pH range 7.0–9.0, while M. luteus 2 and M. lylae immobilized CA showed 80% stability between the pH range 8.0–9.0 and 7.0–7.5, respectively. The temperature stability profile for immobilized CA from P. fragi showed above 80% residual activity between the temperature
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Table 3 Comparison of enzyme activity (EU/g of beads), protein content (g/g beads), specific activity (U/g) and immobilization potential (%) of purified M. luteus 2 carbonic anhydrase. Immobilization period
Immobilization matrix Chitosan–NaOH (B-1)
2h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 4h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 6h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 8h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 10 h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g)
Chitosan–KOH (B-2)
Chitosan–NH4 OH (B-3)
Sodium alginate–CaCl2 (B-4)
Chitosan alginate–CaCl2 (B-5)
Multilayered beads (B-6)
38.43 0.0018 21.35 (35)
48.2 0.0020 24.1 (40)
44.08 0.0019 23.2 (39)
11.7 0.001 11.7 (20)
11.60 0.001 11.6 (19)
10.8 0.0009 12.0 (18)
53.76 0.0021 25.6 (42)
87.90 0.0030 29.3 (59)
74.25 0.0027 27.5 (54)
22.20 0.0014 15.86 (27)
25.65 0.0015 17.1 (29)
25.2 0.0014 18.0 (27)
153.72 0.0036 42.7 (71)
176.28 0.0039 45.2 (78)
130.68 0.0033 39.6 (65)
33.18 0.0016 20.74 (32)
44.08 0.0019 23.2 (39)
45 0.0018 25.0 (36)
182.45 0.0041 44.5 (81)
215.04 0.0042 51.1 (84)
173.66 0.0038 45.7 (75)
44.27 0.0019 23.3 (38)
45.20 0.002 22.6 (40)
52.0 0.002 26.0 (39)
182.45 0.0041 44.5 (81)
215.04 0.0042 51.1 (84)
173.66 0.0038 45.7 (75)
44.27 0.0019 23.2 (38)
45.20 0.002 22.6 (40)
52.0 0.002 26 (39)
The enzyme activity, protein content and specific activity for free purified enzyme were 307.7 (EU), 0.005 (g) and U (100%), respectively. The values within the parentheses indicate the % immobilization. All the errors were within 5% of SD.
Table 3a Comparison of enzyme activity (EU/g of beads), protein content (g/g beads), specific activity (U/g) and immobilization potential (%) of whole cell M. luteus 2 (carbonic anhydrase). Immobilization period
Immobilization matrix Chitosan–NaOH (B-1)
2h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 4h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 6h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 8h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g) 10 h Enzyme activity (EU/g) Protein content (g/g) Specific activity (U/g)
Chitosan–KOH (B-2)
Chitosan–NH4 OH (B-3)
Sodium alginate–CaCl2 (B-4)
Chitosan alginate–CaCl2 (B-5)
Multilayered beads (B-6)
9.93 0.0043 2.31 (41)
5.54 0.0031 1.79 (32)
12.42 0.0046 2.70 (48)
1.38 0.0015 0.92 (16)
1.42 0.0015 0.95 (16)
2.00 0.0018 1.10 (19)
11.33 0.0044 2.58 (46)
5.95 0.0032 1.86 (33)
14.75 0.0050 2.95 (52)
1.70 0.0017 1.00 (18)
1.96 0.0018 1.09 (19)
3.10 0.0023 1.35 (24)
17.98 0.0055 3.27 (57.9)
6.80 0.0034 2.00 (35.4)
17.93 0.0055 3.26 (57.8)
2.18 0.0019 1.15 (20.4)
2.38 0.0020 1.19 (21.1)
3.65 0.0025 1.46 (25.8)
17.98 0.0055 3.27 (57.9)
8.06 0.0037 2.18 (38.6)
25.74 0.0066 3.90 (69.1)
5.64 0.0031 1.82 (32.2)
5.37 0.0030 1.79 (31.7)
6.14 0.0032 1.92 (34)
18.76 0.0056 3.33 (59)
8.06 0.0037 2.18 (38.6)
26.46 0.0067 3.95 (70)
5.95 0.0032 1.86 (33)
6.04 0.0032 1.89 (33.5)
7.31 0.0035 2.09 (37)
The enzyme activity, protein content and specific activity for unimmobilized culture were 53.9 (EU), 0.00955 (g) and 5.64 U (100%) respectively. The values within the parentheses indicate the % immobilization. All the errors were within 5% of SD.
Table 4 Effect of pH and temperature on stability of immobilized CA on Chitosan–KOH beads. Immobilized CA (3 h)
P. fragi M. lylae M. luteus 2
Temperature (◦ C) (residual activity %)
pH (residual activity %) 7.0
7.5
87 (86) 89 (87) 69 (69)
93 (92) 88 (88) 78 (78)
8.0 100 (99) 72 (80) 83 (83)
8.5 90 (85) 68 (68) 91 (90)
9.0 84 (78) 60 (57) 89 (88)
35
40
87 (80) 81 (75) 94 (90)
84 (77) 79 (70) 92 (86)
45 80 (72) 76 (67) 91 (85)
50
55
74 (66) 70 (60) 87 (80)
68 (59) 64 (54) 84 (76)
The values within the parentheses indicate the residual activity of free enzyme (Sharma and Bhattacharya [13]). All the errors were within 5% of SD.
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Table 5 CO2 sequestration efficiency of CA immobilized on chitosan–KOH beads and evaluation of reusability potential in presence of CO2 saturated ASW+ SOx + NOx. The Ca2+ ion concentration was determined after precipitation (5 min). 35 ◦ C 0.2 mM
Control (without enzyme)
45 ◦ C 0.4 mM
Free enzyme
Single cycle
P. fragi CA (% efficiency for calcium utilization & mM of calcium ions utilized)
M. lylae CA (% efficiency for calcium utilization & mM of calcium ions utilized)
M. luteus 2 CA (% efficiency for calcium utilization & mM of calcium ions utilized)
35 ◦ C
45 ◦ C
35 ◦ C
45 ◦ C
35 ◦ C
45 ◦ C
38 [3.8]
53 [5.3]
34.5 [3.45]
48.8 [4.1]
31 [3.1]
44 [4.0]
Reusability cycles
01 02 03 04 05 06 07 08 09 10 Total mM of calcium ion utilized
Immobilized enzyme P. fragi CA (% efficiency for calcium utilization & mM of calcium ions utilized)
M. lylae CA (% efficiency for calcium utilization & mM of calcium ions utilized)
M. luteus 2 CA (% efficiency for calcium utilization & mM of calcium ions utilized)
35 ◦ C
45 ◦ C
35 ◦ C
45 ◦ C
35 ◦ C
45 ◦ C
25 [2.5] 25 [2.5] 18 [1.8] 15 [1.5] 10 [1.0] 07 [0.7] 03 [0.3] – – –
33 [3.3] 33 [3.3] 20 [2.0] 17 [1.7] 13 [1.3] 09 [0.9] 05 [0.5] – – –
19.5 [1.95] 19.5 [1.95] 14 [1.4] 12 [1.2] 08 [0.8] 05 [0.5] – – – –
26 [2.6] 26 [2.6] 17 [1.7] 15 [1.5] 10 [1.0] 07 [0.7] – – – –
17 [1.7] 17 [1.7] 12 [1.2] 10 [1.0] 06 [0.6] 02 [0.2] – – – –
23 [2.3] 23 [2.3] 16 [1.6] 14 [1.4] 09 [0.9] 05 [0.5] – – – –
10.3
13
1.8
10.1
6.4
8.0
Initial calcium concentration for each cycle was 10 mM. The values within the parentheses indicate concentration of calcium ions utilized. All the errors were within 5% SD.
range 35–45 ◦ C, while from M. luteus 2, 80% stability was recorded between 35 and 55 ◦ C. The immobilized M. lylae CA retained above 70% residual activity in the temperature range 35–50 ◦ C.
0.0667), M. lylae (0.0354; 0.0473) and M. luteus 2 (0.0302; 0.0406). During reusability 100% sequestration efficiency was retained for 2 cycles followed by gradual loss in sequestration potential.
3.3. Storage stability
3.5. FTIR analysis
The specific activities on day 1 for free CA from P. fragi, M. lylae and M. luteus 2 were determined as 70.6 U, 66.5 U and 61.0 U respectively. However, the specific activities after day 30 for free CA from P. fragi, M. lylae and M. luteus 2 were determined as 28.9 U, 27.5 U and 22.1 U, respectively. In contrast, the specific activities on day 1 for immobilized CA from P. fragi, M. lylae and M. luteus 2 were determined to be 62.8 U/g beads, 55.2 U/g beads and 51.42 U/g beads, respectively. Similarly, the specific activities on day 30 for immobilized CA from P. fragi, M. lylae and M. luteus 2 were determined to be 52.1 U/g beads, 45.8 U/g beads and 42.4 U/g beads respectively. The storage stability experiments showed that only 41% of residual activity is retained by free CA compared to 83% by immobilized CA.
The FTIR spectra indicated different peaks for chitosan at 3469 cm−1 , 3279 cm−1 , 3192 cm−1 , 3086 cm−1 , 2881 cm−1 , 2870 cm−1 , 1663 cm−1 and 1421 cm−1 (Fig. 1A). Similarly for chitosan–KOH, different peaks were obtained at 3679 cm−1 , 3261 cm−1 , 3190 cm−1 , 2924 cm−1 , 2862 cm−1 , 1678 cm−1 , and 1500 cm−1 (Fig. 1B). The FTIR spectra for free enzyme showed major peaks at 3284 cm−1 , 3072 cm−1 , 2980 cm−1 , 2929 cm−1 , 2879 cm−1 , 1674 cm−1 , 1647 cm−1 , 1527 cm−1 , 1521 cm−1 , and 1460 cm−1 (Fig. 2A) Correspondingly, the spectra for enzyme immobilized chitosan–KOH beads showed major peaks at 3749 cm−1 , 3421 cm−1 , 3275 cm−1 , 2949 cm−1 , 2858 cm−1 , 1683 cm−1 , 1523 cm−1 , 1521 cm−1 , and 1460 cm−1 (Fig. 2B).
3.4. CO2 sequestration efficiency and reusability potential
3.6. XRD analysis
Table 5 shows the percentage efficiency of calcium ion utilization as a measure of CO2 sequestration using immobilized CA from all the three strains. The immobilized CA from P. fragi showed sequestration potential up to 7 recycles, while immobilized CA from M. lylae and M. luteus 2 were effective for 6 recycles only. The total concentration (mM) of calcium ion utilization by 1 g of chitosan–KOH immobilized CA at 35 ◦ C and 45 ◦ C was determined for P. fragi (10.3 mM; 13 mM), M. lylae (7.8 mM; 10.1 mM), and M. luteus 2 (6.4 mM; 9.0 mM). Table 6 shows the total amount of CaCO3 (g) formed following reusability of immobilized CA at 35 ◦ C and 45 ◦ C for P. fragi (0.111; 0.152); M. lylae (0.081; 0.108) and M. luteus 2 (0.069; 0.093). Similarly the gram equivalent of CO2 present in CaCO3 at 35 ◦ C and 45 ◦ C was also calculated for P. fragi (0.0485;
Fig. 3 illustrates the X-ray diffraction pattern obtained for biomimetically produced CaCO3 . The major peaks at 2 = 29.4◦ and 27.08◦ were obtained. The other high intensity peaks were recorded at 2 = 27.1◦ , 32.7◦ , 24.9◦ , 43.8◦ . 3.7. SEM analysis The scanning electron micrographs of calcium carbonate formed by the enzyme are shown in Fig. 4. The image displayed hexagonal shaped crystals. Most of the crystals were of faceted rhombohedral shaped (calcite), however some crystals with spherical characteristics (vaterite) were also visible.
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Table 6 Conversion of CO2 (gram equivalent) into calcium carbonate using CA immobilized on chitosan–KOH beads in presence of CO2 saturated ASW + SOX + NOX. The total amount of calcium carbonate formed was determined after precipitation (5 min). 35 ◦ C 0.2 mM
Control (without enzyme)
45 ◦ C 0.4 mM
Free enzyme P. fragi CA
Single cycle
M. lylae CA
M. luteus 2 CA
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
0.042
0.0184
0.051
0.024
0.032
0.0140
0.041
0.0180
0.029
0.012
0.030
0.0167
Reusability cycles
Immobilized enzyme P. fragi CA
M. lylae CA
M. luteus 2 CA
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
CaCO3
CO2
01 02 03 04 05 06 07
0.027 0.027 0.018 0.016 0.012 0.008 0.003
0.0118 0.0118 0.0079 0.0070 0.0052 0.0035 0.0013
0.035 0.035 0.027 0.021 0.018 0.010 0.006
0.0154 0.0154 0.0118 0.0092 0.0079 0.0044 0.0026
0.020 0.020 0.016 0.012 0.009 0.004 –
0.0088 0.0088 0.0070 0.0052 0.0039 0.0017 –
0.029 0.029 0.018 0.014 0.010 0.006 –
0.0127 0.0127 0.0079 0.0079 0.0044 0.0026 –
0.018 0.018 0.013 0.010 0.007 0.003 –
0.0079 0.0079 0.0057 0.0044 0.0030 0.0013 –
0.024 0.024 0.017 0.014 0.009 0.005 –
0.0105 0.0105 0.0074 0.0061 0.0039 0.0022 –
Total amount of calcium carbonate formed and gram equivalent of CO2 present
0.111
0.0485
0.152
0.0667
0.081
0.0354
0.106
0.0482
0.069
0.0302
0.093
0.0406
All the errors were within 5% SD.
4. Discussion In the present study the maximum specific activity and immobilization potential for immobilized CA from P. fragi (62.8 U/g beads; 89%), M. lylae (55.2 U/g beads; 83%) and M. luteus 2 (51.2 U/g beads; 84%) was obtained on chitosan–KOH. The chitosan derived beads; showed higher immobilization efficiency compared to alginate derived bead and multilayered beads for CA from all the three isolates. Chitosan has hydrophilic, hydroxyl and amino groups that probably facilitate the adsorption of enzyme on the matrix, whereas in alginate or multilayered beads, these groups are either absent or masked leading to less adsorption of enzyme on the matrix and correspondingly lower immobilization efficiency. The findings of Prabhu et al. [15] also substantiated our results. Successful cell immobilization was achieved on all the six materials. Maximum specific activity and efficiency for P. fragi (2.7 U/g beads; 44%) was obtained on multilayered beads. Contrastingly maximum specific activity and efficiency was obtained for M. lylae (3.76 U/g beads; 74%) and M. luteus 2 (3.95 U/g beads; 70%) on chitosan–NH4 OH. The differential adsorption of Gram negative P. fragi and Gram positive M. lylae and M. luteus on chitosan derived beads (chitosan–NaOH, chitosan–KOH and chitosan–NH4 OH) and alginate derived beads is of significant importance. Chitosan is a heteropolymer of glucosamine and acetyl glucosamine units and has many hydrophilic groups. The cell wall of Gram positive bacteria consists mostly of peptidoglycan which is a polymer of N-acetyl glucosamine and N-acetyl muramic acid, amino acids and techoic acid along with surface exposed carbohydrates [21]. Thus, it could be rationally suggested that presence of hydrophilic groups on surface of Gram positive bacteria and chitosan derived beads (chitosan–NaOH, chitosan–KOH and chitosan–NH4 OH) facilitated the bacterial adsorption on these matrix. However, sodium alginate–CaCl2 , chitosan–alginate–CaCl2 and multilayered beads have a more hydrophobic character associated with them thereby non-facilitating the binding of Gram positive bacteria. The higher immobilization efficiency of P. fragi on multilayered beads and alginate beads can be justified from the fact that Gram nega-
tive bacteria have an outer membrane consisting of phospholipids and lipopolysaccharide which imparts a hydrophobic character to these bacteria [22]. The adsorption is thus facilitated by hydrophobic interaction between P. fragi multilayered and alginate derived beads. The study thus substantiated the choice of proper matrix for immobilization of cells and enzymes and corroborated the importance of designing and synthesis of different materials based on the type of sample to be immobilized. The pH profile for immobilized CA from P. fragi, M. lylae and M. luteus 2 was similar to that of the free enzyme. However, there was slight increase in stability of all the three immobilized CA at pH 8.0–9.0. The temperature stability of the immobilized CA showed marked improvement compared to free CA. A maximum increase of 1.08–1.18 fold stability between 35–55 ◦ C for immobilized CA was observed for M. lylae followed by P. fragi (1.08–1.15) and M. luteus 2 (1.04–1.10) compared to free CA. The operational stability of the immobilized CA at elevated temperature has high degree of functional and economical significance associated with it. Bond et al. [2] had already reported the optimum pH range of 8.0–9.0 and temperature range of 35–45 ◦ C as vital components for successful biomimetic sequestration process. The storage stability of immobilized CA in aqueous medium was 2.02 fold higher compared to free CA. This acts as added incentive for the bulk production and storage of immobilized enzyme. The peaks at 3469 cm−1 and 3679 cm−1 for chitosan flakes and chitosan–KOH indicated the stretching vibrations, while the peaks at 3279 cm−1 and 3281 cm−1 for chitosan flakes and chitosan–KOH indicated the O–H stretching vibrations. The N–H and O–H bonds play an important role in hydrophilic interactions like hydrogen bonding and Vander walls interaction, similarly the FTIR spectrum of free enzyme represents the N–H, O–H and C–H stretching vibrations at 3700 cm−1 , 3284 cm−1 and 3072 cm−1 respectively that could be involved in the hydrophilic interactions. The FTIR spectra of CA immobilized on chitosan–KOH also showed the presence of peaks at 3479 cm−1 and 3421 cm−1 corresponding to stretching vibrations due to N–H and O–H groups respectively. The C O stretching at 2924 cm−1 and 2862 cm−1 in chitosan flakes
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Fig. 1. FTIR spectra of (A) chitosan flakes and (B) chitosan–KOH beads.
and chitosan KOH beads was deemed important for hydrogen bonding as they were also identified in free enzyme (2960 cm−1 , 2929 cm−1 and 2379 cm−1 ) and immobilized CA beads (2924 cm−1 and 2858 cm−1 ). The distinct peaks in free enzyme at 1527 cm−1 and 1521 cm−1 correspond to N–H bending vibrations due to presence of amides and/or secondary amines; while peak at 1460 cm−1 indicated the presence of aromatic ring with low degree of substitution (could be attributed to the presence of aromatic amino acids in the enzyme). The FTIR spectra of immobilized enzyme are characterized by N–H bending peaks at 1523 cm−1 and 1512 cm−1 and aromatic ring substitution at 1460 cm−1 thus establishing the successful immobilization of carbonic anhydrase on chitosan–KOH beads. The presence of O–H and N–H groups both on enzyme and immobilized beads confirmed the adsorption due to hydrophilic interactions. At 35 ◦ C, 70.6 U of free P. fragi CA, 66.5 U of free M. lylae CA, and 61.0 U of free M. luteus 2 CA effectively sequestered, 3.8 mM, 3.45 mM, and 3.11 mM respectively, of calcium ions into calcium carbonate. However, using the same U/g immobilized CA from P. fragi, M. lylae, and M. luteus 2 on chitosan–KOH beads sequestered, 10.3 mM, 7.8 mM and 6.4 mM respectively, of calcium ions into calcium carbonate. The reusability of immobilized enzyme allows its repeated use compared to the free enzyme which is lost after single cycle. Thus for any concentration of immobilized CA corresponding
to same concentration of free CA, the amount of CO2 sequestered will always be high. In the present study, the amount of CaCO3 formed and gram equivalent of CO2 present in CaCO3 was 2 fold higher by immobilized CA compared to free CA from all the three strains. Similar phenomenon was also observed with the utilization of calcium ions. The highest sequestration efficiency (∼2.7 fold) was achieved with immobilized CA from P. fragi. Increase in sequestration efficiency at 45 ◦ C was evident for both free and immobilized enzyme. The fact that modest heating (∼50 ◦ C) overcomes inhibition of precipitation [4] in artificial sea water has been proved beyond doubt [13]. These results are very encouraging and advocate the implementation of immobilized enzyme systems at on-site scrubber. A constant loss in sequestration efficiency was observed during reusability of immobilized enzyme. This phenomenon is attributable to the inhibition of enzyme activity due to the presence of different anions in ASW and/or gradual loss of enzyme from the matrix, the latter however needs experimental explication. In an on-site scrubber SOx and NOx are considered to be major inhibitory ions. During our previous study [13], we found that free CA from P. fragi, M. lylae, and M. luteus 2 retained 83%, 92%, and 60% respectively of their residual activity at 0.1 M concentration of sulphate ions, however considerable inhibition was observed for free CA from P. fragi (30%), M. lylae (27%) and M. luteus
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Fig. 2. FTIR spectra of (A) enzyme (carbonic anhydrase) and (B) CA immobilized on chitosan–KOH beads.
(56%) at 0.1 M concentration of nitrate ions. Also Cl− (200 mM), Br− (50 mM), I− (50 mM), and HCO3 − (50 mM), were found to have significant inhibitory effect on all the three CAs. The inhibition profile of each ion varied with CA from three isolates [13]. The calcite phase was found to be the major form of CaCO3 obtained from biomimetic CO2 sequestration using indigenous immobilized CA. The diffraction peaks at 2 = 29.4◦ and 43.8◦ cor-
Fig. 3. XRD spectra of biomimetically produced CaCO3 .
responded to the calcite phase. The other major peaks at 27.08◦ , 27.14◦ and 32.7◦ corresponded to the vaterite phase of CaCO3 . Similar results have been reported by Favre et al. [20] and Li et al. [23]. The SEM images confirmed the presence of both vaterite and calcite phase. A hexagonal structure is obtained with both the phases [24,25], however complex packing in vaterite is illustrated with the spherical appearance of the crystal. The calcite crystals displayed well defined faceted rhombohedral characteristics. In the present study calcite was found to be the most dominant form as it is thermodynamically more stable compared to metastable vaterite at pH < 10 (sequestration was carried out at pH 9.5), however vaterite is dominant above pH 10 [20]. Both Favre et al. [20] and Li et al. [23] have shown that calcite phase is dominant when the precipitation occurs in presence of CA. Phase transformation in crystallographic structure of solid particles with time is a phenomena commonly associated with solid particles grown in liquid medium [26]. Three different mechanisms advocate such solid transformation. The first mechanism involves change from nonuclear mode to either polynuclear mode or mononuclear mode, which was not observed in the present study. The second mechanism of ageing involves dissolution of first burst particle followed by reprecipitation to a more stable calcite phase with time. The third mechanism combines ageing with hydrolysis of products in presence of side chains of proteins [26,27]. Since the protein concentration is very low and the precipitation
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Fig. 4. (A–D) SEM images of biomimetically produced CaCO3 , showing both the calcite and vaterite phases using P. fragi CA immobilized on chitosan–KOH beads.
reaction was initiated in chamber without enzyme, this mechanism seems unlikely. The phase transition is evident in Fig. 4B which clearly shows the highly porous nature of vaterite particles, while the thin planar non-porous crystals of calcite are visible, surfacing from the vaterite particles, thus supporting the dissolution reprecipitation mechanism of ageing. CA accelerates the formation of bicarbonate ions by lowering the activation energy required for hydration of CO2 . The conversion of CO2 into H2 CO3 is the rate controlling step catalyzed by CA, while the formation of HCO3 − is nearly diffusion controlled [6]. However, the conversion of HCO3 − to CO3 2− is pH dependent [4,20]. An enzyme can only increase the rate of the reaction, but could not alter it, thus CA is deemed to accelerate the formation of bicarbonate ions. Under the precipitation conditions (pH 9.0–9.5) bicarbonate ions exist in equilibrium with carbonate ions and in presence of calcium ions result in calcium carbonate precipitation. Thus CA plays the important role in hydration reaction corresponding to calcium carbonate formation, calcite being the dominant form [20]. Thus application of an effective immobilization system operating at low mass (protein) values under process parameters will open up a new avenue for cost effective sequestration of CO2 into CaCO3 in an onsite scrubber.
5. Conclusions The study highlighted the effectiveness of different immobilization matrices corresponding to both Gram positive and Gram negative bacteria. The study for the first time reports the biomimetic sequestration of CaCO3 using immobilized CA from three indigenous strains under conditions simulating an on-site scrubber. The CO2 sequestration potential and reusability of immobilized CA compared to free CA provides this system an immense advantage and towering edge along with economic relevance and commercial utility.
Acknowledgements The authors are grateful to DBT, New Delhi for providing the financial assistance. A. Bhattacharya is thankful to Council of Scientific and Industrial Research (CSIR), New Delhi for providing the CSIR-SRF fellowship. The FTIR facility provided by Dr. Anjali Bajpai, Department of Chemistry, Govt. Model Science College, Jabalpur (M.P.) and XRD and SEM facility by Dr. Sadhna Rayalu, Head EMD, National Environmental Engineering Research Institute (NEERI), Nagpur is highly acknowledged. The authors are also thankful to the Head, Department of Biological Science, Rani Durgavati University, Jabalpur (M.P.), for providing laboratory facilities.
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