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Enzyme and Microbial Technology 48 (2011) 416–426

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

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iomimetic CO2 sequestration using purified carbonic anhydrase fromndigenous bacterial strains immobilized on biopolymeric materials

njana Sharma ∗, Abhishek Bhattacharya, Ankita Shrivastavaacteriology Laboratory, Department of P.G. Studies and Research in Biological Science, Rani Durgavati University, Pachpedi, Jabalpur 482001, Madhya Pradesh, India

r t i c l e i n f o

rticle history:eceived 3 October 2010eceived in revised form 2 February 2011ccepted 2 February 2011

a b s t r a c t

The present study deals with immobilization of purified CA and whole cell of Pseudomonas fragi, Micro-coccus lylae, and Micrococcus luteus 2 on different biopolymer matrices. Highest enzyme immobilizationwas achieved with P. fragi CA (89%) on chitosan–KOH beads, while maximum cell immobilization wasachieved with M. lylae (75%) on chitosan–NH4OH beads. A maximum increase of 1.08–1.18 fold stability

eywords:iomimeticO2-sequestration

mmobilizationarbonic anhydrase

between 35 and 55 ◦C was observed for M. lylae immobilized CA. The storage stability was improvedby 2.02 folds after immobilization. FTIR spectra confirmed the adsorption of CA on chitosan–KOH beadsfollowing hydrophilic interactions. Calcium carbonate precipitation was achieved using chitosan–KOHimmobilized P. fragi CA. More than 2 fold increase in sequestration potential was observed for immobilizedsystem as compared to free enzyme. XRD spectra revealed calcite as the dominant phase in biomimetically

ate.
. fragindigenous
produced calcium carbon

. Introduction

The rising concentration of green house gases (GHGs), and inarticular carbon dioxide (CO2) due to anthropogenic interven-ions has led to several undesirable consequences such as globalarming and related changes. The most easily addressable source

f CO2 is that from the generation of electric power, and partic-larly that from the coal burning stations, since they comprise aelatively small number of very large stationary sources [1]. Coalred power plants currently account for little more than half thelectricity generation and it is unlikely that there will be a dramatichange in this situation in the next twenty years [2,3].

Conversion of CO2 into solid carbonates offers the possibility ofsafe and stable ecofriendly product for long term carbon seques-

ration [2]. Precipitation from aqueous solution occurs at a suitableupersaturation of calcium and carbonate ions [4]. The hydration ofO2 to form carbonic acid is the rate-limiting step in the conversionf CO2 into carbonate ions, which has a forward reaction constantf 6.2 × 10−3 s at 25 ◦C [5,6]. This hydration reaction is catalyzed byarbonic anhydrase (CA) at or near diffusion controlled limit [7].
arbonic anhydrase (CA) is one of the fastest enzymes that cataly-es CO2-hydration reaction with typical rates between 104 and 106
eactions per second for different forms of this enzyme [8]. It is ainc metalloenzyme reported to be present in animals, plants and

∗ Corresponding author. Tel.: +91 761 2416667; fax: +91 761 2603752;obile: +91 9425155323.

E-mail address: [email protected] (A. Sharma).

141-0229/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2011.02.001

© 2011 Elsevier Inc. All rights reserved.

microorganisms [9–12]. The successful precipitation of CO2 (aq)into CaCO3 in the presence of calcium ions through biomimeticapproach involving, indigenous carbonic anhydrase [13] and com-mercial Bovine Carbonic anhydrase (BCA) [14] has been proved inprinciple. However, commercial application of this approach war-rants the immobilization of CA on to a suitable immobilizationmatrix for application at an onsite scrubber [14].

The process of immobilization confines enzymes/whole cellsto a phase distinct from the one in which the substrates and theproducts are present [15]. Chitosan and sodium alginate are inertmaterials that have been used for immobilization of enzymes andmicroorganism [15]. Immobilization enhances the efficiency of theprocess by allowing reuse of enzyme coupled with easy removalof the products from the reaction mixture. The physical meth-ods especially adsorption, have an advantage over the chemicalmethods for immobilization of enzymes onto carriers in that it issimple, less expensive and can retain high catalytic activity [16].CO2 sequestration using immobilized BCA and indigenous CA underdifferent process parameters is under research phase [13,15] and anobjectively defined study involving indigenous bacterial CA immo-bilized on inert matrix is the need of the hour.

The present study thus aims at immobilization of both CA andbacterial cells of indigenous origin on chitosan and alginate basedmaterials and to assess their CO2 sequestration potential.

2. Materials and methods

Artificial sea water (ASW) and chitosan were purchased from Sigma–Aldrich, StLouis, MO, USA. Sodium alginate was purchased from Himedia Mumbai, India. All thebuffers used in the study were purchased from Himedia Mumbai, India. The sodium

dx.doi.org/10.1016/j.enzmictec.2011.02.001

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dx.doi.org/10.1016/j.enzmictec.2011.02.001

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nd potassium salts of chloride, bromide, iodide, fluoride, carbonate, sulphate anditrate were purchased from Sisco Research Laboratory, Mumbai, India.

.1. Synthesis of materials

Chitosan–NaOH beads (B-1), chitosan–KOH beads (B-2), chitosan–NH4OH beadsB-3), sodium alginate–CaCl2 beads (B-4), chitosan–sodium alginate–CaCl2 beadsB-5) and multilayered beads (B-6), were prepared following the method of Prabhut al. [15].

.2. Source of the bacterial strain and carbonic anhydrase

The indigenous purified CA from Pseudomonas fragi (BGCC /= 1077), Micrococcusylae (BGCC /= 1078), and Micrococcus luteus 2 (BGCC /= 1079) were obtained fromacterial Germplasm Collection Centre (BGCC), Bacteriology Laboratory, Depart-ent of P.G. Studies and Research in Biological Science, R.D. University, Jabalpur

M.P.), India.

.3. Immobilization

.3.1. Immobilization of enzymeLyophilized CA (5 mg) was dissolved in 5 ml of 50 mM Tris–HCl (pH 8.0) and

ixed separately with 1 g of the immobilization matrix. The experimental setupontaining 3 sets was designed. Each set with five separate tubes contained enzymemmobilization matrix suspension for P. fragi CA, M. lylae CA and M. luteus 2 CA, sep-rately. The suspension in all the three sets was agitated (120 rpm) at 4 ◦C and themmobilization efficiency was determined at every 2 h interval up to 10th h, usingsingle tube containing the suspension each from the three sets designated for CA

rom the three indigenous strains. After immobilization the beads were separatedrom the suspension and enzyme activity (EU) and protein concentration (mg) inhe supernatant was determined. The direct estimation of CO2 hydration activity oneads after immobilization was carried out using 200 mg immobilized enzyme. Theotal enzyme activity on the beads was also determined as the difference betweenhe total enzyme activity of free enzyme in solution before immobilization and theotal enzyme activity present in the solution after immobilization. The total enzymectivity of immobilized CA was expressed as EU/g beads and the mass transfer ofnzyme on immobilized beads was determined as amount of protein retained oneads after immobilization i.e. difference in protein concentration before and after

mmobilization in the suspension and was expressed as g protein/g beads. The spe-ific activity (U) [where, U = EU/mg protein], on the immobilized beads was alsoetermined and was expressed as U/g beads. The immobilization potential (%) wasetermined in terms of specific activity retained on beads compared to that of freenzyme (100%).

.3.2. Immobilization of microorganismsCulture (100 ml) of each strain (A600 nm = 1.2) and 1 g of different immobiliza-

ion matrices was added into flask and incubated at 37 ◦C for 10 h with 2 h intervalt 120 rpm. After incubation the slurry was centrifuged and the beads were col-ected, washed thoroughly with sterile distilled water and suspended in Tris–HCluffer (50 mM, pH 8.0). The sample was sonicated thrice for 10 s with 30 s inter-als followed by centrifugation at 10,000 rpm for 10 min [15]. Similarly the cultureA600 nm = 1.2) without any immobilization matrix was centrifuged and suspendedn Tris–HCl buffer (50 mM, pH 8.0), followed by sonication and centrifugation asescribed above. The supernatant obtained was used to determine the enzyme activ-

ty (EU) and protein concentration (mg). The total enzyme activity of immobilizedells was expressed as EU/g beads and the mass transfer of cells on immobilizedeads was determined as amount of protein retained on beads after immobilizationnd was expressed as g protein/g beads. The specific activity (U) [where, U = EU/mgrotein], on the immobilized beads was also determined and was expressed as U/geads. The immobilization potential (%) was determined in terms of specific activityetained on beads compared to that of free culture (100%).

.4. Determination of carbonic anhydrase activity

The method of Sharma and Bhattacharya [13] was followed with certain mod-fications. 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 water3 ml) was immediately added to 2 ml of Tris–HCl buffer (100 mM; pH 8.3) [13], and.1 ml of free purified CA (1 mg/ml stock), 0.1 ml of supernatant left after the immo-ilized enzyme was separated from suspension and 0.1 ml of supernatant derivedfter sonication of cell culture and immobilized cells were transferred immediately
ut separately to determine the enzyme activity. The time required for the pH change.0–7.0 (t) was measured. The time required for the pH change (8.0–7.0) was useds control (tc), when buffer was substituted for test sample. The enzyme assay wasarried out at 4 ◦C. The Wilbur Anderson units were calculated with the equationtc − t)/t [17]. The protein content was determined by the method of Lowry et al.18].
l Technology 48 (2011) 416–426 417

2.5. Effect of pH and temperature on immobilized CA

The stability of the CA immobilized on chitosan–KOH beads was assayed usingtwo different buffer systems, 50 mM phosphate buffer (pH 7.0, 7.5 and 8.0) andTris–HCl buffer (pH 8.5 and 9.0) at 37 ◦C for 3 h. The temperature stability of immo-bilized CA on chitosan–KOH beads was determined by carrying out the enzymereactions at different temperatures (35–55 ◦C) and pH 8.0 for 3 h. The enzyme activ-ity at the start of the experiment was taken as 100% and the residual activity wasdetermined after incubation.

2.6. Storage stability

The storage stability of both the free enzyme and immobilized enzyme wasdetermined by storing them for 30 days at 4 ◦C. The residual activity was determinedfollowing 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 anhydraseand CA immobilized on chitosan–KOH beads mixed with KBr pellets were recordedon Shimadzu FTIR spectrophotometer (V-110). Spectra of all the materials werescanned in the range of 400–4000 cm−1.

2.8. Determining CO2 sequestration efficiency using immobilized carbonicanhydrase

CO2 saturated solution using artificial sea water (ASW) was prepared at roomtemperature as described under Section 2.4. ASW was supplemented with K2SO4

(0.1 M) and KNO3 (0.1 M) to study the effect of SOx and NOx on CA activity andsequestration efficiency. CO2 saturated solution (10 ml) was mixed with 1 ml of Trisbuffer (pH 8.3) containing free CA (100 �g) from all three bacterial strains separatelyfor 15 min. The bicarbonate solution was released into another vessel through a valvecontaining 10 ml of CaCl2 solution (at a final concentration of 10.0 mM). To the abovemixture, Tris-buffer pH 9.5 (2 ml, 1 M) was immediately added. The reaction mix-ture was incubated at 35 ◦C and 45 ◦C, respectively for 5 min to allow precipitationof CaCO3 and the amount of CaCO3 formed was determined [13]. The control exper-iment was carried out in the absence of enzyme and the results were expressedin terms of mg CaCO3 formed following control correction. CA from all the threeindigenous isolates immobilized on chitosan–KOH beads was used for sequestrationstudies by replacing the free enzyme in the reaction chamber.

The sequestration efficiency was evaluated by determining the ionic concen-tration of calcium present before and after carbonate precipitation following themethod of Kolthof et al. [19]. The difference in calcium concentration was consideredto be the amount of calcium utilized in formation of calcium carbonate. An enzymefree system was used as control for comparison. The percentage efficiency of cal-cium ion utilized was also calculated and results were reported following controlcorrection. The weight of calcium carbonate was also calculated and gm equivalentsof 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. Thereaction of CO2 saturated solution with immobilized enzyme was carried out ina chamber separate from the vessel used for CaCO3 precipitation. The CA beadswere 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 beingconnected through a two way valve allows its easy removal, washing of beads andrefining into the main apparatus for further reuse.

2.10. XRD analysis

The X-ray diffraction spectra of biomimetically produced CaCO3 were recordedon a Rigaku Miniflex II instrument using Cu K� radiation (� = 0.15406 nm) operatedat 30 kV and 15 mA.

2.11. SEM analysis

Scanning electron microscopy was performed on selected carbonate samplesgenerated biomimetically. A JEOL JSM 5800 LV electron microscope under an elec-trical tension of 20 kV was used for this purpose. A small solid piece of each sample tobe examined was placed on a sample holder covered with a carbon tab and metalizedwith 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 determinedfollowing CO2 hydration assay.

418 A. Sharma et al. / Enzyme and Microbial Technology 48 (2011) 416–426

Table 1Comparison 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)

Chitosan–KOH(B-2)

Chitosan–NH4OH(B-3)

Sodiumalginate–CaCl2(B-4)

Chitosan alginate–CaCl2(B-5)

Multilayeredbeads (B-6)

2 hEnzyme activity (EU/g) 46.98 62.16 56.4 12.06 13.90 11.7Protein content (g/g) 0.0018 0.0021 0.002 0.0009 0.0001 0.0009Specific activity (U/g) 26.1 (37) 29.6 (42) 28.2 (40) 13.4 (19) 13.9 (20) 13 (18)

4 hEnzyme activity (EU/g) 88.25 120.64 102.87 24.83 28.56 28Protein content (g/g) 0.0025 0.0029 0.0027 0.0013 0.0014 0.0014Specific activity (U/g) 35.3 (50) 41.6 (59) 38.1 (54) 19.1 (27) 20.4 (29) 20 (28)




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he enzyme activity, protein content and specific activity for free purified enzyme wndicate the % immobilization. All the errors were within 5% of SD.

.1. Screening of materials for immobilization of enzyme and cells

Determination of enzyme and cell immobilization in terms ofpecific activity showed that maximum CA immobilization waschieved after 8 h of incubation; while maximum cell immobiliza-ion was achieved following 10 h of incubation irrespective of theype of immobilization material used.

Maximum immobilization of CA from all the three
trains was obtained on B-2 followed by B-3 and B-1,ess than 50% immobilization was achieved with B-4, B-
and B-6 materials (Table 1, Table 2, and Table 3). CAmmobilized on chitosan–KOH (B-2) was used for further

able 1aomparison of enzyme activity (EU/g of beads), protein content (g/g beads), specific activi



Chitosan–KOH(B-2)

Chito(B-3)

2 hEnzyme activity (EU/g) 1.42 0.19 0.36Protein content (g/g) 0.0016 0.0006 0.000Specific activity (U/g) 0.89 (15) 0.32 (5.3) 0.45

4 hEnzyme activity (EU/g) 2.58 0.40 0.57Protein content (g/g) 0.0021 0.0008 0.001Specific activity (U/g) 1.23 (20) 0.48 (7.8) 0.57

6 hEnzyme activity (EU/g) 3.10 1.04 1.32Protein content (g/g) 0.0023 0.0013 0.001Specific activity (U/g) 1.35 (22) 0.80 (13) 0.88



he enzyme activity, protein content and specific activity for unimmobilized culture werendicate the % immobilization. All the errors were within 5% of SD.

3 (EU), 0.005 (g) and 70.6 U (100%) respectively. The values within the parentheses

studies. The immobilization potential (%), enzyme activity(EU/g beads) and mass transfer (g/g beads) were determinedfor CA from all the three strains (Table 1, Table 2, and Table 3).Maximum mass transfer (g/g beads) and enzyme activity (EU/gbeads) 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/gbeads; 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 observedon B-2. However, maximum cell immobilization for M. lylae andM. luteus 2 was observed on B-3 followed by B-1 and B-2. Min-

ty (U/g) and immobilization potential (%) of whole cell P. fragi (carbonic anhydrase).

san–NH4OH Sodiumalginate–CaCl2(B-4)

Chitosanalginate–CaCl2(B-5)


3.17 3.36 3.408 0.0023 0.0024 0.0024

(7.3) 1.38 (22) 1.40 (23) 1.42 (23)

3.70 3.28 4.410 0.0025 0.0024 0.0027

(9.3) 1.48 (24) 1.37 (22) 1.63 (26)

9.28 6.73 9.765 0.0040 0.0034 0.0041

(14) 2.32 (38) 1.98 (32) 2.38 (39)

11.79 6.73 12.697 0.0045 0.0034 0.0047

(16) 2.62 (43) 1.98 (32) 2.70 (44)

11.79 7.45 12.697 0.0045 0.0036 0.0047

(16) 2.62 (43) 2.07 (34) 2.70 (44)

64.5 (EU), 0.01057 (g), and U (100%) respectively. The values within the parentheses

A. Sharma et al. / Enzyme and Microbial Technology 48 (2011) 416–426 419

Table 2Comparison 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.



Chitosan–KOH(B-2)





2 hEnzyme activity (EU/g) 41.94 53.2 49.4 3.32 10.8 9.90Protein content (g/g) 0.0018 0.002 0.0019 0.0005 0.0009 0.0009Specific activity (U/g) 23.3 (35) 26.6 (40) 26.0 (39) 6.65 (10) 12.0 (18) 11.0 (17)


6 hEnzyme activity (EU/g) 153.68 169.75 169.46 30 41.9 41.4Protein content (g/g) 0.0034 0.0035 0.0037 0.0015 0.0018 0.0018Specific activity (U/g) 45.2 (68) 48.5 (69) 45.8 (73) 20.0 (30) 23.3 (35) 23.0 (35)



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 parenthesesindicate the % immobilization. All the errors were within 5% of SD.

Table 2aComparison 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).



Chitosan–KOH(B-2)










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he enzyme activity, protein content and specific activity for unimmobilized cultarentheses indicate the % immobilization. All the errors were within 5% of SD.

mum immobilization was observed on B-4 (Table 1a, Table 2a,nd Table 3a). The immobilization potential (%), enzyme activityEU/g beads) and mass transfer (g/g beads) were determined forree cells (Table 1a, Table 2a and Table 3a). Maximum mass transfermg/mg beads) and enzyme activity (EU/g beads) were observed onhitosan–NH4OH (B-3) beads for M. luteus 2 cell (0.0067 g/g beads;6.46 EU/g beads), followed by M. lylae (0.0074 mg/mg beads;
7.12 EU/beads) cell and on multi-layered beads (B-6) for P. fragiell (0.0047 g/g beads; 12.69 EU/g beads). Since only a maximum of5% cell immobilization (M. lylae on B-3) was obtained compared to9% enzyme immobilization of CA from P. fragi (B-2), immobilizedells were not considered for further study.
ere 50.1 (EU), 0.00988 (g) and 5.07 U (100%) respectively. The values within the

3.2. Effect of pH and temperature on stability of immobilized CA

The stability of free and immobilized CA at different pH andtemperature is illustrated in Table 4. The results show that at pH8.0, 100% activity was retained for immobilized P. fragi CA, while72% and 83% residual activity was retained for M. lylae and M. luteus2 immobilized CA respectively. The immobilized CA from P. fragi
showed 80% stability in the pH range 7.0–9.0, while M. luteus 2and M. lylae immobilized CA showed 80% stability between the pHrange 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


Table 3Comparison 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.



Chitosan–KOH(B-2)







6 hEnzyme activity (EU/g) 153.72 176.28 130.68 33.18 44.08 45Protein content (g/g) 0.0036 0.0039 0.0033 0.0016 0.0019 0.0018Specific activity (U/g) 42.7 (71) 45.2 (78) 39.6 (65) 20.74 (32) 23.2 (39) 25.0 (36)



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 parenthesesindicate the % immobilization. All the errors were within 5% of SD.

Table 3aComparison 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 (carbonicanhydrase).

Immobilization period Immobilization matrixChitosan–NaOH(B-1)

Chitosan–KOH(B-2)







6 hEnzyme activity (EU/g) 17.98 6.80 17.93 2.18 2.38 3.65Protein content (g/g) 0.0055 0.0034 0.0055 0.0019 0.0020 0.0025Specific activity (U/g) 3.27 (57.9) 2.00 (35.4) 3.26 (57.8) 1.15 (20.4) 1.19 (21.1) 1.46 (25.8)

8 hEnzyme activity (EU/g) 17.98 8.06 25.74 5.64 5.37 6.14Protein content (g/g) 0.0055 0.0037 0.0066 0.0031 0.0030 0.0032Specific activity (U/g) 3.27 (57.9) 2.18 (38.6) 3.90 (69.1) 1.82 (32.2) 1.79 (31.7) 1.92 (34)

10 hEnzyme activity (EU/g) 18.76 8.06 26.46 5.95 6.04 7.31Protein content (g/g) 0.0056 0.0037 0.0067 0.0032 0.0032 0.0035Specific activity (U/g) 3.33 (59) 2.18 (38.6) 3.95 (70) 1.86 (33) 1.89 (33.5) 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 theparentheses indicate the % immobilization. All the errors were within 5% of SD.

Table 4Effect of pH and temperature on stability of immobilized CA on Chitosan–KOH beads.

Immobilized CA (3 h) pH (residual activity %) Temperature (◦C) (residual activity %)

7.0 7.5 8.0 8.5 9.0 35 40 45 50 55

P. fragi 87 (86) 93 (92) 100 (99) 90 (85) 84 (78) 87 (80) 84 (77) 80 (72) 74 (66) 68 (59)M. lylae 89 (87) 88 (88) 72 (80) 68 (68) 60 (57) 81 (75) 79 (70) 76 (67) 70 (60) 64 (54)M. luteus 2 69 (69) 78 (78) 83 (83) 91 (90) 89 (88) 94 (90) 92 (86) 91 (85) 87 (80) 84 (76)

T
he values within the parentheses indicate the residual activity of free enzyme (Sharma a nd Bhattacharya [13]). All the errors were within 5% of SD.


Table 5CO2 sequestration efficiency of CA immobilized on chitosan–KOH beads and evaluation of reusability potential in presence of CO2 saturated ASW+ SOx + NOx. The Ca2+ ionconcentration was determined after precipitation (5 min).

Control (without enzyme) 35 ◦C 45 ◦C0.2 mM 0.4 mM

Free enzyme

P. fragi CA (% efficiency for calciumutilization & mM of calcium ionsutilized)

M. lylae CA (% efficiency for calciumutilization & mM of calcium ionsutilized)

M. luteus 2 CA (% efficiency for calciumutilization & mM of calcium ionsutilized)

35 ◦C 45 ◦C 35 ◦C 45 ◦C 35 ◦C 45 ◦C

Single cycle 38 [3.8] 53 [5.3] 34.5 [3.45] 48.8 [4.1] 31 [3.1] 44 [4.0]

Reusability cycles Immobilized enzyme

P. fragi CA (% efficiency for calciumutilization & mM of calcium ionsutilized)

M. lylae CA (% efficiency for calciumutilization & mM of calcium ionsutilized)

M. luteus 2 CA (% efficiency forcalcium utilization & mM ofcalcium ions utilized)

35 ◦C 45 ◦C 35 ◦C 45 ◦C 35 ◦C 45 ◦C

01 25 [2.5] 33 [3.3] 19.5 [1.95] 26 [2.6] 17 [1.7] 23 [2.3]02 25 [2.5] 33 [3.3] 19.5 [1.95] 26 [2.6] 17 [1.7] 23 [2.3]03 18 [1.8] 20 [2.0] 14 [1.4] 17 [1.7] 12 [1.2] 16 [1.6]04 15 [1.5] 17 [1.7] 12 [1.2] 15 [1.5] 10 [1.0] 14 [1.4]05 10 [1.0] 13 [1.3] 08 [0.8] 10 [1.0] 06 [0.6] 09 [0.9]06 07 [0.7] 09 [0.9] 05 [0.5] 07 [0.7] 02 [0.2] 05 [0.5]07 03 [0.3] 05 [0.5] – – – –08 – – – – – –09 – – – – – –10 – – – – – –

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Total mM of calcium ion utilized 10.3 13

nitial calcium concentration for each cycle was 10 mM. The values within the pare

ange 35–45 ◦C, while from M. luteus 2, 80% stability was recordedetween 35 and 55 ◦C. The immobilized M. lylae CA retained above0% residual activity in the temperature range 35–50 ◦C.

.3. Storage stability

The specific activities on day 1 for free CA from P. fragi, M. lylaend M. luteus 2 were determined as 70.6 U, 66.5 U and 61.0 U respec-ively. However, the specific activities after day 30 for free CA from. fragi, M. lylae and M. luteus 2 were determined as 28.9 U, 27.5 Und 22.1 U, respectively. In contrast, the specific activities on day 1or immobilized CA from P. fragi, M. lylae and M. luteus 2 were deter-

ined to be 62.8 U/g beads, 55.2 U/g beads and 51.42 U/g beads,espectively. Similarly, the specific activities on day 30 for immo-ilized CA from P. fragi, M. lylae and M. luteus 2 were determined toe 52.1 U/g beads, 45.8 U/g beads and 42.4 U/g beads respectively.he storage stability experiments showed that only 41% of residualctivity is retained by free CA compared to 83% by immobilized CA.

.4. CO2 sequestration efficiency and reusability potential

Table 5 shows the percentage efficiency of calcium ion utiliza-ion as a measure of CO2 sequestration using immobilized CA fromll the three strains. The immobilized CA from P. fragi showedequestration potential up to 7 recycles, while immobilized CArom M. lylae and M. luteus 2 were effective for 6 recycles only.he total concentration (mM) of calcium ion utilization by 1 g ofhitosan–KOH immobilized CA at 35 ◦C and 45 ◦C was determinedor P. fragi (10.3 mM; 13 mM), M. lylae (7.8 mM; 10.1 mM), and M.
uteus 2 (6.4 mM; 9.0 mM). Table 6 shows the total amount of CaCO3g) formed following reusability of immobilized CA at 35 ◦C and5 ◦C for P. fragi (0.111; 0.152); M. lylae (0.081; 0.108) and M. luteus(0.069; 0.093). Similarly the gram equivalent of CO2 present in
aCO3 at 35 ◦C and 45 ◦C was also calculated for P. fragi (0.0485;

1.8 10.1 6.4 8.0

s indicate concentration of calcium ions utilized. All the errors were within 5% SD.

0.0667), M. lylae (0.0354; 0.0473) and M. luteus 2 (0.0302; 0.0406).During reusability 100% sequestration efficiency was retained for 2cycles followed by gradual loss in sequestration potential.

3.5. FTIR analysis

The FTIR spectra indicated different peaks for chitosan at3469 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 forchitosan–KOH, different peaks were obtained at 3679 cm−1,3261 cm−1, 3190 cm−1, 2924 cm−1, 2862 cm−1, 1678 cm−1, and1500 cm−1 (Fig. 1B). The FTIR spectra for free enzyme showedmajor 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, and1460 cm−1 (Fig. 2A) Correspondingly, the spectra for enzymeimmobilized chitosan–KOH beads showed major peaks at3749 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).

3.6. XRD analysis

Fig. 3 illustrates the X-ray diffraction pattern obtained forbiomimetically produced CaCO3. The major peaks at 2� = 29.4◦ and27.08◦ were obtained. The other high intensity peaks were recordedat 2� = 27.1◦, 32.7◦, 24.9◦, 43.8◦.

3.7. SEM analysis

The scanning electron micrographs of calcium carbonate formedby the enzyme are shown in Fig. 4. The image displayed hexagonalshaped crystals. Most of the crystals were of faceted rhombohedralshaped (calcite), however some crystals with spherical character-istics (vaterite) were also visible.


Table 6Conversion of CO2 (gram equivalent) into calcium carbonate using CA immobilized on chitosan–KOH beads in presence of CO2 saturated ASW + SOX + NOX. The total amountof calcium carbonate formed was determined after precipitation (5 min).

Control (without enzyme) 35 ◦C 45 ◦C0.2 mM 0.4 mM

Free enzyme

P. fragi CA M. lylae CA M. luteus 2 CA

CaCO3 CO2 CaCO3 CO2 CaCO3 CO2 CaCO3 CO2 CaCO3 CO2 CaCO3 CO2

Single cycle 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 0.027 0.0118 0.035 0.0154 0.020 0.0088 0.029 0.0127 0.018 0.0079 0.024 0.010502 0.027 0.0118 0.035 0.0154 0.020 0.0088 0.029 0.0127 0.018 0.0079 0.024 0.010503 0.018 0.0079 0.027 0.0118 0.016 0.0070 0.018 0.0079 0.013 0.0057 0.017 0.007404 0.016 0.0070 0.021 0.0092 0.012 0.0052 0.014 0.0079 0.010 0.0044 0.014 0.006105 0.012 0.0052 0.018 0.0079 0.009 0.0039 0.010 0.0044 0.007 0.0030 0.009 0.003906 0.008 0.0035 0.010 0.0044 0.004 0.0017 0.006 0.0026 0.003 0.0013 0.005 0.002207 0.003 0.0013 0.006 0.0026 – – – – – – – –

Total amount ofcalcium carbonateformed and gramequivalent of CO

0.111 0.0485 0.152 0.0667 0.081 0.0354 0.106 0.0482 0.069 0.0302 0.093 0.0406

A

4

l88sdlpimcP

rbm(cfbaaatoaTgbiahnia

2

present

ll the errors were within 5% SD.

. Discussion

In the present study the maximum specific activity and immobi-ization potential for immobilized CA from P. fragi (62.8 U/g beads;9%), M. lylae (55.2 U/g beads; 83%) and M. luteus 2 (51.2 U/g beads;4%) was obtained on chitosan–KOH. The chitosan derived beads;howed higher immobilization efficiency compared to alginateerived bead and multilayered beads for CA from all the three iso-

ates. Chitosan has hydrophilic, hydroxyl and amino groups thatrobably facilitate the adsorption of enzyme on the matrix, whereas

n alginate or multilayered beads, these groups are either absent orasked leading to less adsorption of enzyme on the matrix and

orrespondingly lower immobilization efficiency. The findings ofrabhu et al. [15] also substantiated our results.

Successful cell immobilization was achieved on all the six mate-ials. Maximum specific activity and efficiency for P. fragi (2.7 U/geads; 44%) was obtained on multilayered beads. Contrastinglyaximum 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%) onhitosan–NH4OH. The differential adsorption of Gram negative P.ragi and Gram positive M. lylae and M. luteus on chitosan derivedeads (chitosan–NaOH, chitosan–KOH and chitosan–NH4OH) andlginate derived beads is of significant importance. Chitosan isheteropolymer of glucosamine and acetyl glucosamine units

nd has many hydrophilic groups. The cell wall of Gram posi-ive bacteria consists mostly of peptidoglycan which is a polymerf N-acetyl glucosamine and N-acetyl muramic acid, amino acidsnd techoic acid along with surface exposed carbohydrates [21].hus, it could be rationally suggested that presence of hydrophilicroups on surface of Gram positive bacteria and chitosan derivedeads (chitosan–NaOH, chitosan–KOH and chitosan–NH4OH) facil-

tated the bacterial adsorption on these matrix. However, sodium
lginate–CaCl2, chitosan–alginate–CaCl2 and multilayered beadsave a more hydrophobic character associated with them therebyon-facilitating the binding of Gram positive bacteria. The higher
mmobilization efficiency of P. fragi on multilayered beads andlginate beads can be justified from the fact that Gram nega-

tive bacteria have an outer membrane consisting of phospholipidsand lipopolysaccharide which imparts a hydrophobic character tothese bacteria [22]. The adsorption is thus facilitated by hydropho-bic interaction between P. fragi multilayered and alginate derivedbeads. The study thus substantiated the choice of proper matrix forimmobilization of cells and enzymes and corroborated the impor-tance of designing and synthesis of different materials based on thetype 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 wasslight increase in stability of all the three immobilized CA at pH8.0–9.0. The temperature stability of the immobilized CA showedmarked improvement compared to free CA. A maximum increaseof 1.08–1.18 fold stability between 35–55 ◦C for immobilized CAwas 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 stabil-ity of the immobilized CA at elevated temperature has high degreeof functional and economical significance associated with it. Bondet al. [2] had already reported the optimum pH range of 8.0–9.0 andtemperature range of 35–45 ◦C as vital components for successfulbiomimetic sequestration process. The storage stability of immo-bilized CA in aqueous medium was 2.02 fold higher compared tofree CA. This acts as added incentive for the bulk production andstorage of immobilized enzyme.

The peaks at 3469 cm−1 and 3679 cm−1 for chitosan flakes andchitosan–KOH indicated the stretching vibrations, while the peaksat 3279 cm−1 and 3281 cm−1 for chitosan flakes and chitosan–KOHindicated the O–H stretching vibrations. The N–H and O–H bondsplay an important role in hydrophilic interactions like hydrogenbonding and Vander walls interaction, similarly the FTIR spec-trum of free enzyme represents the N–H, O–H and C–H stretchingvibrations at 3700 cm−1, 3284 cm−1 and 3072 cm−1 respectively
that could be involved in the hydrophilic interactions. The FTIRspectra of CA immobilized on chitosan–KOH also showed thepresence of peaks at 3479 cm−1 and 3421 cm−1 corresponding tostretching 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


n flak

ab2aaeisacasbii

a3cf1crc

Fig. 1. FTIR spectra of (A) chitosa

nd chitosan KOH beads was deemed important for hydrogenonding as they were also identified in free enzyme (2960 cm−1,929 cm−1 and 2379 cm−1) and immobilized CA beads (2924 cm−1

nd 2858 cm−1). The distinct peaks in free enzyme at 1527 cm−1

nd 1521 cm−1 correspond to N–H bending vibrations due to pres-nce of amides and/or secondary amines; while peak at 1460 cm−1

ndicated the presence of aromatic ring with low degree of sub-titution (could be attributed to the presence of aromatic aminocids in the enzyme). The FTIR spectra of immobilized enzyme areharacterized by N–H bending peaks at 1523 cm−1 and 1512 cm−1

nd aromatic ring substitution at 1460 cm−1 thus establishing theuccessful immobilization of carbonic anhydrase on chitosan–KOHeads. The presence of O–H and N–H groups both on enzyme and

mmobilized beads confirmed the adsorption due to hydrophilicnteractions.

At 35 ◦C, 70.6 U of free P. fragi CA, 66.5 U of free M. lylae CA,nd 61.0 U of free M. luteus 2 CA effectively sequestered, 3.8 mM,.45 mM, and 3.11 mM respectively, of calcium ions into calciumarbonate. However, using the same U/g immobilized CA from P.
ragi, M. lylae, and M. luteus 2 on chitosan–KOH beads sequestered,0.3 mM, 7.8 mM and 6.4 mM respectively, of calcium ions into cal-ium carbonate. The reusability of immobilized enzyme allows itsepeated use compared to the free enzyme which is lost after singleycle. Thus for any concentration of immobilized CA corresponding
es and (B) chitosan–KOH beads.

to same concentration of free CA, the amount of CO2 sequesteredwill always be high. In the present study, the amount of CaCO3formed and gram equivalent of CO2 present in CaCO3 was 2 foldhigher by immobilized CA compared to free CA from all the threestrains. Similar phenomenon was also observed with the utilizationof calcium ions. The highest sequestration efficiency (∼2.7 fold) wasachieved with immobilized CA from P. fragi. Increase in sequestra-tion efficiency at 45 ◦C was evident for both free and immobilizedenzyme. The fact that modest heating (∼50 ◦C) overcomes inhi-bition of precipitation [4] in artificial sea water has been provedbeyond doubt [13]. These results are very encouraging and advo-cate the implementation of immobilized enzyme systems at on-sitescrubber.

A constant loss in sequestration efficiency was observed dur-ing reusability of immobilized enzyme. This phenomenon isattributable to the inhibition of enzyme activity due to the pres-ence of different anions in ASW and/or gradual loss of enzymefrom the matrix, the latter however needs experimental explica-tion. In an on-site scrubber SOx and NOx are considered to be
major inhibitory ions. During our previous study [13], we foundthat 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 con-centration of sulphate ions, however considerable inhibition wasobserved for free CA from P. fragi (30%), M. lylae (27%) and M. luteus


drase)

((no

oi

Fig. 2. FTIR spectra of (A) enzyme (carbonic anhy

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 sig-

ificant inhibitory effect on all the three CAs. The inhibition profilef each ion varied with CA from three isolates [13].

The calcite phase was found to be the major form of CaCO3btained from biomimetic CO2 sequestration using indigenousmmobilized CA. The diffraction peaks at 2� = 29.4◦ and 43.8◦ cor-

Fig. 3. XRD spectra of biomimetically produced CaCO3.

and (B) CA immobilized on chitosan–KOH beads.

responded to the calcite phase. The other major peaks at 27.08◦,27.14◦ and 32.7◦ corresponded to the vaterite phase of CaCO3. Sim-ilar results have been reported by Favre et al. [20] and Li et al. [23].The SEM images confirmed the presence of both vaterite and cal-cite phase. A hexagonal structure is obtained with both the phases[24,25], however complex packing in vaterite is illustrated withthe spherical appearance of the crystal. The calcite crystals dis-played well defined faceted rhombohedral characteristics. In thepresent study calcite was found to be the most dominant formas it is thermodynamically more stable compared to metastablevaterite at pH < 10 (sequestration was carried out at pH 9.5), how-ever vaterite is dominant above pH 10 [20]. Both Favre et al.[20] and Li et al. [23] have shown that calcite phase is dominantwhen the precipitation occurs in presence of CA. Phase transfor-mation in crystallographic structure of solid particles with timeis a phenomena commonly associated with solid particles grownin liquid medium [26]. Three different mechanisms advocate suchsolid transformation. The first mechanism involves change fromnonuclear mode to either polynuclear mode or mononuclear mode,which was not observed in the present study.

The second mechanism of ageing involves dissolution of firstburst particle followed by reprecipitation to a more stable cal-cite phase with time. The third mechanism combines ageing withhydrolysis of products in presence of side chains of proteins [26,27].Since the protein concentration is very low and the precipitation


e calc

rnwwfr

tsC[[cbbpTifowi

5

bGbtsbac

Fig. 4. (A–D) SEM images of biomimetically produced CaCO3, showing both th

eaction was initiated in chamber without enzyme, this mecha-ism seems unlikely. The phase transition is evident in Fig. 4Bhich clearly shows the highly porous nature of vaterite particles,hile the thin planar non-porous crystals of calcite are visible, sur-

acing from the vaterite particles, thus supporting the dissolutioneprecipitation mechanism of ageing.

CA accelerates the formation of bicarbonate ions by loweringhe activation energy required for hydration of CO2. The conver-ion of CO2 into H2CO3 is the rate controlling step catalyzed byA, while the formation of HCO3

− is nearly diffusion controlled6]. However, the conversion of HCO3

− to CO32− is pH dependent

4,20]. An enzyme can only increase the rate of the reaction, butould not alter it, thus CA is deemed to accelerate the formation oficarbonate ions. Under the precipitation conditions (pH 9.0–9.5)icarbonate ions exist in equilibrium with carbonate ions and inresence of calcium ions result in calcium carbonate precipitation.hus CA plays the important role in hydration reaction correspond-ng to calcium carbonate formation, calcite being the dominantorm [20]. Thus application of an effective immobilization systemperating at low mass (protein) values under process parametersill open up a new avenue for cost effective sequestration of CO2

nto CaCO3 in an onsite scrubber.

. Conclusions

The study highlighted the effectiveness of different immo-ilization matrices corresponding to both Gram positive andram negative bacteria. The study for the first time reports theiomimetic sequestration of CaCO3 using immobilized CA from
hree indigenous strains under conditions simulating an on-sitecrubber. The CO2 sequestration potential and reusability of immo-ilized CA compared to free CA provides this system an immensedvantage and towering edge along with economic relevance andommercial utility.
[

ite and vaterite phases using P. fragi CA immobilized on chitosan–KOH beads.

Acknowledgements

The authors are grateful to DBT, New Delhi for providing thefinancial assistance. A. Bhattacharya is thankful to Council of Sci-entific and Industrial Research (CSIR), New Delhi for providing theCSIR-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 theHead, Department of Biological Science, Rani Durgavati University,Jabalpur (M.P.), for providing laboratory facilities.

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