recent advancements in carbonic anhydrase–driven processes for co 2 ...

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This article was downloaded by: [University of Chicago Library] On: 29 September 2013, At: 01:29 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Critical Reviews in Environmental Science and Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/best20 Recent Advancements in Carbonic Anhydrase–Driven Processes for CO 2 Sequestration: Minireview Ajam Yakub Shekh a , Kannan Krishnamurthi a , Sandeep N. Mudliar b , Raju R. Yadav a , Abhay B. Fulke a , Sivanesan Saravana Devi a & Tapan Chakrabarti a a Environmental Health Division, CSIR-National Environmental Engineering Research Institute (NEERI), Nagpur, India b Environmental Biotechnology Division, CSIR-National Enviornmental Engineering Research Institute (NEERI), Nagpur, India Accepted author version posted online: 30 Aug 2011.Published online: 25 May 2012. To cite this article: Ajam Yakub Shekh , Kannan Krishnamurthi , Sandeep N. Mudliar , Raju R. Yadav , Abhay B. Fulke , Sivanesan Saravana Devi & Tapan Chakrabarti (2012) Recent Advancements in Carbonic Anhydrase–Driven Processes for CO 2 Sequestration: Minireview, Critical Reviews in Environmental Science and Technology, 42:14, 1419-1440, DOI: 10.1080/10643389.2011.556884 To link to this article: http://dx.doi.org/10.1080/10643389.2011.556884 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

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This article was downloaded by: [University of Chicago Library]On: 29 September 2013, At: 01:29Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Critical Reviews in EnvironmentalScience and TechnologyPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/best20

Recent Advancements in CarbonicAnhydrase–Driven Processes for CO2

Sequestration: MinireviewAjam Yakub Shekh a , Kannan Krishnamurthi a , Sandeep N. Mudliarb , Raju R. Yadav a , Abhay B. Fulke a , Sivanesan Saravana Devi a &Tapan Chakrabarti aa Environmental Health Division, CSIR-National EnvironmentalEngineering Research Institute (NEERI), Nagpur, Indiab Environmental Biotechnology Division, CSIR-National EnviornmentalEngineering Research Institute (NEERI), Nagpur, IndiaAccepted author version posted online: 30 Aug 2011.Publishedonline: 25 May 2012.

To cite this article: Ajam Yakub Shekh , Kannan Krishnamurthi , Sandeep N. Mudliar , Raju R.Yadav , Abhay B. Fulke , Sivanesan Saravana Devi & Tapan Chakrabarti (2012) Recent Advancementsin Carbonic Anhydrase–Driven Processes for CO2 Sequestration: Minireview, Critical Reviews inEnvironmental Science and Technology, 42:14, 1419-1440, DOI: 10.1080/10643389.2011.556884

To link to this article: http://dx.doi.org/10.1080/10643389.2011.556884

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

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Critical Reviews in Environmental Science and Technology, 42:1419–1440, 2012Copyright © Taylor & Francis Group, LLCISSN: 1064-3389 print / 1547-6537 onlineDOI: 10.1080/10643389.2011.556884

Recent Advancements in CarbonicAnhydrase–Driven Processes for CO2

Sequestration: Minireview

AJAM YAKUB SHEKH,1 KANNAN KRISHNAMURTHI,1

SANDEEP N. MUDLIAR,2 RAJU R. YADAV,1 ABHAY B. FULKE,1

SIVANESAN SARAVANA DEVI,1 and TAPAN CHAKRABARTI11Environmental Health Division, CSIR-National Environmental Engineering Research

Institute (NEERI), Nagpur, India2Environmental Biotechnology Division, CSIR-National Enviornmental Engineering Research

Institute (NEERI), Nagpur, India

The authors reviews the advancements in carbonic anhydrase–driven processes for CO2 sequestration research and engineering.Historical and recent discoveries of carbonic anhydrase and ideabehind using it for CO2 sequestration are elaborated as well as theuses of this enzyme in free and immobilized forms are thoroughlydiscussed. New concepts such as extension of immobilized enzymesystems for bioreactor approach with the aim of CO2 abatement atthe source are also introduced briefly toward the end of the review.The authors also suggest the possible future directions to employcarbonic anhydrase for CO2 sequestration.

KEY WORDS: bioreactor, carbonic anhydrase, CO2 sequestration,enzyme immobilization

1. INTRODUCTION

Developing countries in the world are in the process of raising their econ-omy and need more energy. As a result the exploitation of fossil fuels, mostlyhuge coal reserves, is becoming unavoidable, leading to the anthropogenic

Address correspondence to Dr. K. Krishnamurthi, Enviornmental Health Division, CSIR-National Environmental Engineering Research Institute (NEERI), Nehru Marg, Nagpur, 440020,India. Tel: +91-712-2249757; Fax: +91-712-2249961. E-mail: k [email protected]

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emission of major greenhouse gases, mainly carbon dioxide (CO2), methane(CH4), nitrous oxide (N2O), ozone (O3), and chlorofluorocarbons (Intergov-ernmental Panel on Climate Change [IPCC], 2007). These greenhouse gasesare strongly and steadily contributing toward the warming of earth’s surface(Bhattacharyya et al., 2004) by the absorption of infrared radiations.

CO2 is one of the major contributors to the warming of the earth amongall the greenhouse gases. Anthropogenic emission of CO2 is continuously ris-ing all over the world (Keeling et al., 1995). During the decade of 1990–1999,an average increase in atmospheric CO2 level was 1.1% per year, and it roseto over 3% per year from 2000 to 2004. Recent literature envisages that theglobal temperature is expected to rise by 4C by 2100 (Reifert, 2007). Ac-cording to the Intergovernmental Panel on Climate Change (IPCC) report,warming of about 0.1C per decade would be expected even if the concen-trations of entire greenhouse gases and aerosols had been kept constant atthe levels reported for the year 2000 (IPCC, 2007).

It is generally recognized that the visual effects of the constantly increas-ing CO2 concentration on the ¡ earth can be seen in the form of more violentstorms, changes in the course of Gulf stream, increasing temperature of theearth, melting of the polar ice sheets, and elevated sea levels (Sharma et al.,2009). These climatic changes are likely to affect human health with respectto cardiorespiratory disorders and heat wave–related disorders. In addition,psychological and social instability may possibly rise in the affected areas.Geographical distribution of vector organisms such as malaria mosquitoesmay increase because of increase in ambient temperature (McMichael et al.,2006; United Nations Environmental Programme, 1996). Therefore, an im-mediate and considerable reduction in CO2 emission through carbon man-agement is required to prevent a worst case situation pertaining to globalclimate change.

In the past decade, various carbon management strategies have beenemployed and the same can be achieved using three different but compli-mentary approaches such as (a) efficient energy conversion, (b) utilizationof low-carbon or carbon-devoid energy sources, and (c) CO2 capture and itssequestration. It is commonly accepted that the first two options will onlyprovide incremental improvements. Therefore, there is an urgent need to de-velop appropriate and efficient carbon sequestration technologies to reduceCO2 emissions in the environment (Maroto-Valer et al., 2005).

The most common CO2 sequestration approaches include use ofchemical/physical solvents, adsorption onto solids, membranes, cryo-genic/condensation systems (Abu-Khader, 2006), geological sequestration,and deep ocean sequestration. The utilization cost has proven to be highlyexpensive for the previously mentioned approaches (Abu-Khader, 2006).Therefore, scientists are focusing more on the development of economicand sustainable technologies for CO2 sequestration. In this regard, the en-zymatic sequestration of CO2 into the mineral carbonation seems to be onepossible promising approach.

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Sequestration of CO2 to mineral carbonates offers the prospect of asafe, steady, and environmentally friendly product for long-term carbonsequestration (Bond et al., 2005; Favre et al., 2009; Mirjafari et al., 2007;Ramanan et al., 2009a,b), as considerable carbonate mineral reservoirs haveexisted for millions of years, provided the cheap and large-scale appropriatecation supplies (e.g., Ca, Mg) are available. On a geological time frame, CO2

sequestration into mineral carbonates is a laborious and slow process. Inorder to make the reaction faster under a conducive environment, whichprogresses through the hydration of CO2, an enzyme known as carbonicanhydrase (CA) is employed (Favre et al., 2009; Mirjafari et al., 2007).

CA is a metalloenzyme containing zinc (Zn++) metal ion in its active site,encoded by almost all organisms including eukaryotes and prokaryotes, andcatalyzes the reversible hydration of carbon dioxide (CO2) into bicarbonateions (HCO−3 ). This enzyme accelerates CO2 hydration dramatically providedthe pH is above the pKa of CO2/HCO−3 equilibrium. The most active CAhydrates CO2 at rates as high as kcat = 106 s−1, or a million times a second(Berg et al., 2007; Campbell and Reece, 2005). The enzyme has been studiedas a catalyst in both free as well as in immobilized forms.

This review especially focuses on the enzymatic CO2 sequestration ap-proaches, where the CA has been used as a catalyst in free as well as in im-mobilized forms. The unique catalytic property of CA enzyme had resultedin acceleration of CO2 sequestration process much faster than it occurs inthe natural time frame, ultimately making the CO2 sequestration (via mineralcarbonation) processes much quicker, cost-effective, and sustainable.

2. CARBONIC ANHYDRASE: HISTORICAL AND RECENT PROGRESS

CA is an ancient enzyme, widespread among the entire prokaryotic andeukaryotic domain, and has been known to catalyze the reversible hydrationof carbon dioxide (Smith and Ferry, 2000; Tripp et al., 2001) as follows:

[CO2 + H2O↔ HCO−3 + H+]

The discovery of the CA enzyme is attributed to Meldrum and Roughton(1933), who characterized the enzyme for the first time as a result of curiosityfor knowing the factors responsible for the rapid transit of the HCO−3 fromerythrocytes to the pulmonary capillary. Even though CA was discoveredin 1933, the absolute purification was achieved only in the late 1930s frombovine erythrocytes (Keilin and Mann, 1939, 1940, 1944). The entire discov-eries and advancements regarding the fundamental studies of CA were not inthe domain of scientific community working with bioenvironmental sciencesuntil the 1990s.

Keilin and Mann (1939, 1940, 1944) identified that CA is the first zincmetalloenzyme containing zinc metal ions in its active site and Zn ions hasthe specific role in the reaction(s) catalyzed by CA. A +2 charge on Zn ion

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attracts the oxygen of water molecule and thus helps in bringing the waterand carbon dioxide in close proximity with each other. Binding of watermolecules to zinc lowers the pKa of water and promotes removal of proton,leaving zinc bound to a hydroxide ion, which is a strong nucleophile (Berget al., 2007; Campbell and Reece, 2005). Neish (1939) reported that the CAof plant origin has no similarities to previously known classes of CA withrespect to the size. However, this enzyme has shown decreased sensitivityto sulfonamide inhibitors.

Three decades after the discovery of CA in eukaryotes, Veitch andBlankenship (1963) for the first time, reported the CA enzyme in prokaryotes.This enzyme was found to share the properties with human CA. Advance-ments in the prokaryotic CA related studies were very slow and it tookalmost a decade when the first prokaryotic CA was reported to be puri-fied from Neisseria sicca in 1972 (Adler et al., 1972). It took another twodecades for availability of the first report on sequence of CA from microbialorigin. The first sequence of CA in prokaryotes was reported in 1992 fromCA isolated and purified from Escherichia coli (Guilloton et al., 1992).

cynT has been identified as the gene encoding for CA in E. coli andfound to be the active element of cynate operon along with the two othergenes (cynS and cynX). The gene encoding for cynase (cynS) and geneencoding for the protein of unknown function (cynX) collectively catalyzethe cynate conversion to NH+4 and CO2. The first product (NH+4 ) is utilizedas the source of nitrogen and the second product (CO2) is made availablefor the cellular processes and for making HCO−3 accessible for the catalysisof cynate. In the previous reaction, CA plays an important role in catalyzingthe hydration of CO2 to HCO−3 and thus helps in maintaining the cellularfunctioning and prevents the growth inhibition by cynate. This role of CA hasbeen studied by the various gene-specific deletion mutation studies of cynateoperon in E. coli. CA (cynT gene product) has the molecular weight of 24kDa and shown considerable sequence identity with spinach/pea sequencesand, thus, it is regarded as the first β-class CA identified in prokaryotes(Guilloton et al., 1993).

In 1994, Alber and Ferry isolated the first CA from the archea domain.This new CA was placed in γ -class of CAs because it did not show signifi-cant amino acid sequence similarity with any of the two previously identifiedclasses of CA (α and/or β). This γ -class CA was isolated, purified, and se-quenced from methanoarchaeon, Methanosarcina thermophila (Alber andFerry, 1994). Various CAs were identified and purified from different plants,prokaryotic species and other archaeon, and finally conclusions were drawnthat the β-class contributes in all three domains of life; α- and β-class pre-dominates in eukaryotes and γ -class in archaea (Smith and Ferry, 2000; Trippet al., 2001).

Roberts et al. (1997) reported δ-class, a new class of CA. This new class ofzinc metalloenzyme was purified and partially sequenced from T. weissflogii.

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Interestingly, no significant sequence similarity was found when comparedwith the sequence of three known classes of CAs (α, β, γ ) present at thattime. The approximate molecular weight determined by SDS-PAGE of thisenzyme was found to be 27 kDa (Lane and Morel, 2000). Further researchled to the discovery of new classes of CA. Carboxysomal CA, designatedas CsoS3, was purified and sequenced from the carboxysomal shell of thechemolithoautotroph Halothiobacillus neapolitanus. The molecular weightof the enzyme, calculated from its primary structure, was 57.3 kDa. Thisenzyme was found to have two domains as a result of gene duplication andonly one of the domains was characterized for the functional Zn2+ bindingsite. This shell protein was initially classified as the ε-class of CA becauseit showed significant difference in sequence compared to the previouslyknown classes of CAs such as α, β, γ , and δ (Tripp et al., 2001; Anthonyet al., 2004). On the contrary, crystal structure of the same revealed that itis a new subclass of β-CA representing the distinct active site as is confinedto only a single domain of the enzyme, while in other β-CA, pair of activesites is organized within the two different domains. Despite this divergence inactive site confinement, there is remarkable structural similarity among activesite regions of CAs and hence it is suggested that the CAs have commoncatalytical mechanism for the interconversion of HCO−3 and CO2 (Sawayaet al., 2006). Marine cynobacteria Prochlorococcus sp., and Synecoccus sp.also contain these CAs in their carboxysomal shells. There is a diffusionbarrier to gases because of carboxysomal shells. CA in the shell plays animportant role in transporting and converting the HCO−3 into CO2 inside thecarboxysome where the CO2 is used by ribulose biphosphate carboxylase(Rubisco; Heinhorst et al., 2006).

Lane et al. (2005) identified, isolated, and partially characterized a gene(cdca1) from marine diatom Thalassiosira weissflogii. The purified protein(CDCA1) from the previously mentioned diatom was characterized for CAactivity and was found to contain cadmium in its active site in place of zinc(Zn2+). It was identified that this enzyme had not been sequenced beforeand was unique with respect to the amino acid sequence; hence it wascategorized as the new class of CA, ζ -class. The molecular weight of thisenzyme was determined as 69 kDa. The presence of a cadmium bindingsite in the purified protein was confirmed by X-ray absorption near-edgespectroscopy. It was also confirmed that the cadmium ion was bound bytwo or more thiolates with roughly tetrahedral geometry. Lane et al. (2005)also found that the expression of protein CDCA1 can be controlled by theavailability of cadmium and CO2 in seawater.

Three novel classes of CAs (δ, ε, ζ ) have been discovered in recentpast apart from pre-existing three classes of CAs (α, β, γ ). In addition tothis, one of the CA has been placed in subclass of β-CA. Looking at suchbiodiversity, it will not be surprising if some more new classes of CA arediscovered in the near future. Even if some of the newly discovered CAs

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consists of other metals (e.g., cadmium) in their active site instead of Zn2+,they show similar primary structure of the active site and exhibit commoncatalytical mechanism for reversible hydration of CO2, which is described inthe next section.

2.1 Carbonic Anhydrase: Mechanism of Action

The catalytic mechanism of the CAs, especially alpha-CAs, has been exten-sively studied. It follows a two-step isomechanism. From the evidences ithas been suggested that CO2 hydration is initiated by the nucleophilic attackof zinc-bound hydroxide ion on the carbon atom of CO2. In the next step,the enzyme undergoes regeneration of active site by the ionization of zinc-bound water molecule and causes removal of proton from the active site.Active site consists of a Zn (II) ion coordinated by three histidine residuesand a water molecule/hydroxide ion. The latter is the active species, whichacts as a potent nucleophile. Although some β-class enzymes do not havewater directly coordinated to the metal ion, the zinc hydroxide mechanismis also valid for β and γ CAs (Domsic, 2010; Supuran, 2008). Referring toFigure 1, the stepwise catalytical mechanism of CA for hydration of CO2 isexplained as follows:

• A zinc prosthetic group in the enzyme is coordinated in three positionsby histidine side chains. The fourth coordination position is occupied bywater. This causes polarization of the hydrogen-oxygen bond, making theoxygen slightly more negative, thereby weakening the bond.

FIGURE 1. The zinc-bound hydroxide mechanism for the hydration of carbon dioxide cat-alyzed by CA (adapted from Berg et al., 2007; Campbell and Reece, 2005) (Color figureavailable online).

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Carbonic Anhydrase for CO2 Sequestration 1425

• The fourth histidine (not coordinated to Zn) is placed close to the substrateof water (Zn-water ligand) and accepts a proton—an example of generalacid–general base catalysis. This leaves a hydroxide attached to the zinc.• The active site also contains specificity pocket for carbon dioxide, bringing

it close to the hydroxide group. This allows the electron-rich hydroxideto attack the carbon dioxide, forming a bicarbonate ion (Domsic, 2009).

2.2 Carbonic Anhydrase as a Tool for CO2 Sequestration: Concept

The industrial revolution is leading to the emission of CO2, one of the majoranthropogenic greenhouse gases. The concentration of CO2 has increased by40% from the preindustrial level, which is analogous to a concentration risefrom 280 to 360 ppm (Mirjafari et al., 2007). CO2 is the reaction by-productof combustion, without any fuel value, and is of environmental concernbecause it has been identified as the major contributor to the phenomenonof global warming, on which humanity may be able to have an action.Therefore, the issue of reducing the emission of CO2 to the atmosphere is ofserious concern.

Various methods available for reducing the emission and sequestrationof CO2 to the atmosphere are proving costly (Herzog, 2001). An eco-friendlyand cost-effective approach for CO2 sequestration has been developed byexploiting the ability of CA to catalyze the reversible hydration of CO2.Hydration of CO2 releases the bicarbonate ion into the reaction medium,which is then targeted by an appropriate metal ion to precipitate eco-friendlysolid carbonate, which can be stored for millions of years (Bond et al., 2001;Mirjafari et al., 2007; Ramanan et al., 2009a). The hydration reaction of theaqueous CO2 occurs independently without the use of CA. As a result, solidcalcium carbonate is produced in the presence of calcium ion.

The reactions are depicted as follows:

1. The aqueous CO2 and water react to give proton and bicarbonate ion:

CO2 (aq.)+H2Ok1←→k−1

H+ +HCO−3 (1)

Where, K1 = [CO2] / [H+] · [HCO−3 ]

K1 = k1/k−1.

2. Carbonic acid then dissociates to bicarbonate and carbonate ions:

HCO−3 ↔ H+ + CO2−3 (2)

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3. In the presence of Ca2+ cations, CaCO3 precipitates as follows:

Ca2+ + CO2−3 → CaCO3 ↓ (3)

At 37C near neutral pH, the second-order rate constant (K1) is 0.0026 M−1

s−1 where k1 and k-1 values are 6.2 × 10−2 s−1 and 23.7 s−1, respectively.Whereas the equilibrium constant of reaction 2 (K2) is equal to 1.7 × 10−4

(Mirjafari et al., 2007). Among the entire reaction series for the precipitationof CaCO3 (Reactions 1–3), the hydration of CO2 to form carbonate ion andproton (Reaction 1) forwards with an extremely slow pace and is the rate-limiting step. When the biocatalyst CA is employed to catalyze the hydrationof CO2 (Reaction 1), the rate of hydration increases dramatically, making thereaction much faster, and hydrating CO2 at rates as high as 1.4 × 106 M/s−1.Likewise, the CA enzyme plays a major role in making the reaction muchfaster and thus plays a vital role for CO2 sequestration.

2.3 Carbonic Anhydrase: Activity Assay

CA shows two different kinds of activities, hydrase activity and esterase ac-tivity, which can be determined by using different methods. Hydrase activitycan be assayed using electrometric method developed by Wilbur and An-derson in 1948. For this assay, 8 ml ice-cold CO2-saturated water was addedto 12 ml of 20 mM tris buffer (pH 8.3) incubated with enzyme. The changein pH from 8.3 to 6.3 at 4C was monitored and the time required for this2 units pH drop was recorded. Wilbur-Anderson (hereafter WA) activity ofCA was calculated using following formula and expressed as WA units permilligram of protein.

CA activity = [(t0/t− 1) ∗ 20]/mg protein

Wherein coefficient 20 is the dilution factor for CA, and to and t signifythe time required for 2 units pH drop from 8.3 to 6.3 in control and intest sample, respectively (Zhang et al., 2009). Different groups of scientificworkers have used the WA method with slight alterations in the parameterssuch as enzyme concentration and the final volume of reaction mediumwith same ratio of buffer to CO2-saturated water. However, critical analysisof studies reported by Mirjafari et al. (2007) and Bond et al. (2001), by EkremOzdemir (2009) showed that the data obtained from CO2 hydration couldbring large uncertainties.

It was hard to carry out the activity assay of CA with CO2 as gaseoussubstrate. Therefore, the esterase activity of the enzyme was measured inliquid phase with para nitrophenol acetate (p-NPA) as a substrate and theproduction of para nitro phenol (p-NP) was monitored at 25C. The CAactivity was carried out in 1 ml UV cuvette. The reaction mixture consisted of

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0.05 M 800 µl tris buffer of pH 7.5, to which were added 100 µl of substrate(p-NPA dissolved in acetonitrile) solution and 100 µl of enzyme solution.Following quick and appropriate mixing of solution, the absorbance of p-NPwas measured at wavelength of 400 nm. However; p-NPA undergoes selfdissociation depending on concentration and temperature. Hence the rate ofthe self dissociation (which obeys first-order kinetics) has to be subtractedfrom the enzyme-catalyzed reaction rate to report the esterase activity of CA(Ozdemir, 2009).

3. BIOMIMETIC CO2 SEQUESTRATION: FREE/SOLUBLECARBONIC ANHYDRASE

The pioneering work in CA-driven biomimetic CO2 sequestration has beencarried out by Bond et al. (2001). The same scientific group (Liu et al., 2005)used synthetic brines with compositions similar to those of produced watersfrom oil and gas production in the Permian and San Juan Basins as potentialsources for cations (Ca2+ and Mg2+) in biomimetic carbonate formation. Thestudies were carried out by analyzing the entire precipitation reaction. Thereaction parameters such as time for precipitate formation, concentration ofCa2+ before and after precipitation, and the concentration of CO2 as totalinorganic carbon (IC) were monitored. At the same time, efficiency of CO2

sequestration was also estimated as a percent change in total concentrationof IC in solution after precipitation. Fifty-four percent efficiency of CO2

sequestration was reported for Permian basin samples, which was high ascompared with 13% sequestration efficiency attributed to samples of SanJuan Basin because the concentration of Ca2+ (143 mg) and HCO−3 (467 mg)was comparatively more than San Juan Basin sample, which had Ca2+ (84mg) and HCO−3 (804 mg) (Liu et al., 2005; Bond et al., 2004).

Mirjafari et al. (2007) investigated the role of CA for CO2 hydrationby studying CO2 mineralization as one of the methods for converting CO2

to mineral carbonate. Experiments involved the bubbling of CO2 gas intodeionized water to prepare CO2-saturated water. Reaction medium consistedof 15 ml of phosphate buffer and 5 ml of enzyme solution. Reaction startedwith the addition of 20 ml CO2-saturated water in to the reaction medium.Bovine CA (BCA) was used with varying concentrations (0.2, 0.4, 0.6, 0.8, 1,2, and 6 µM) as well as temperatures (0, 30, and 50C), and the influenceof both on CO2 hydration and precipitation of CaCO3 was studied. BCA wasfound to enhance the rate of hydration of CO2, leading to the rapid initiationof precipitaion of calcium carbonate (CaCO3). At constant temperature andhigh concentration of enzyme, the rate of CO2 hydration was maximum. Onthe contrary, the increasing temperature of reaction medium led to decreasein CO2 hydration rate. Less amount of CaCO3 was found to be precipitated athigher temperatures and settled quickly in presence of an enzyme. Formation

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of CaCO3 was also found to be affected by the pH of reaction medium(Mirjafari et al., 2007).

Favre et al. (2009) studied the formation kinetics of CaCO3 at temper-atures of 5 and 20C with buffers having two different pHs (8.5 and 10.5).The total reaction volume was 27 ml despite the variations in the volumeof individual constituents such as buffer, CO2-saturated water, and enzymesolution (BCA). Then, 0.3 g of CaCl2.2H2O was used as a constant source forthe Ca2+ ions. With a buffer solution of pH 10.5 at 5C, the formation rate ofCaCO3 was found to increase more than 10 times with BCA. The study alsoconcluded that the concentration of enzyme and buffer capacity of the reac-tion medium play important role in increasing the formation rate of HCO−3 .Otherwise, the pH of reaction medium drops so quickly that in the end itmay not favor the precipitation of CaCO3 and, as a consequence, the pre-cipitation of solid carbonate may be extremely hindered. Precipitated CaCO3

was characterized by X-ray diffraction (XRD), Fourier transformed infraredspectroscopy (FTIR), and scanning electron microcopy (SEM). From scan-ning electron micrographs of the solid CaCO3 it has been observed that twodifferent phases of CaCO3, vaterite and calcite, have been formed dependingon the reaction conditions used. At 5C with pH 10.5 and without enzyme inthe reaction medium, CaCO3 was precipitated as vaterite whereas at lowerpH, or at elevated temperature (20C), and in the presence of enzyme inreaction medium calcite was precipitated (Favre et al., 2009).

Our earlier research work (Ramanan et al., 2009a,b), for the first time,reported the isolation, purification, and sequencing of CA from enterobacterspecies, Citrobacter freundii and Bacillus subtilis. The molecular weight ofboth the enzymes have been confirmed by SDS-PAGE and found to be 24and 37 kDa for the polypeptides from C. freundii and B. subtilis, respectivelyThe enzymes from B. subtilis and C. freundii were subjected to varioustemperature and pH to ascertain the extent of their stability. The enzymefrom B. subtilis was found to be stable over the pH range of 7.0–11.0 andwas optimally active at pH 8.3. An isoelectric point (pI) of around 6.1 wasconfirmed by isoelectric focusing (IEF) of the purified enzyme (Ramananet al., 2009b). The enzyme retained nearly 100% activity at pH 8.0 and 8.3,compared with 70% activity recorded at pH 7.0 and 9.0 and, at pH 11.0, only20% activity was observed. Enzyme was found to be stable at a temperaturerange until the upper maximum of 60C while optimal activity was at 37C.Further, enzyme kinetics was performed for CA enzyme of B. subtilis. Vmax,Km, and Ki values were measured using Lineweaver-Burk plots of CA activityat optimal pH and temperature using various concentrations of p-NPA as asubstrate. The Km of CA was 9.09 mM and the Vmax was 714.28 µmol min−1

mg−1. The Ki values for acetazolamide and sulfanilamide were 0.22 and0.099 mM, respectively. Later, both the enzymes (as published in differentstudies) were employed for a lab-scale CO2 sequestration approach. Thestudy of the effect of various concentrations of CO2 and the inhibitor(s)

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Carbonic Anhydrase for CO2 Sequestration 1429

was also carried out. A wide range of inhibitors for CA and those chemicalspecies that contribute to the flue gas composition were chosen for the study,SO−4 , NO−2 , NO−3 , EDTA, and acetazolamide. The enzyme was also incubatedwith 1 mM salt solution of metal ions such as Ca2+, Co2+, Cu2+, fe3+, Hg2+,Mg2+, Mn2+, Pb2+, and Zn2+. As discussed in the experimental section, thereaction was initiated by the addition of free CA to the reaction mixture ofCO2-saturated calcium chloride solution of pH 8.3. Carbonate/bicarbonatedeposition by purified enzyme was found to be 225 mg CO3

2−/HCO−3 permilligram of protein. The study showed that the crude enzyme was almost15 times less active than the purified enzyme.

The metal ions such as Pb2+ and Hg2+ were found to inhibit the CAactivity. Ca2+ and Mn2+ were noted as the weak inhibitors and observed thatit may not affect an enzyme for CO2 sequestration. Apart from zinc metal ion,Co2+, Cu2+, and Fe3+ were found to act as the enhancers of enzyme activity.Anions such as Cl−, HCO−3 , and CO2−

3 were found as strong inhibitors of CA.SO2−

4 , the major component of flue gas, was the only anion found to activateCA and is contradictory to the results reported by Bond et al. (2001), whichshows SO2−

4 acts as inhibitor to CA (Ramanan et al., 2009a; Ramanan et al.,2009b).

Anjana Sharma and Abhishek Bhattacharya (2010) studied the enhancedbiomimetic CO2 sequestration into CaCO3. CAs isolated and purified fromthree different bacteria Pseudomonas fragi (PCA), Micrococcus luteus 2(MTCA), and Micrococcus lylae (MLCA) were used in the study along withcommercial BCA. WA activities for all the enzymes were determined. Effectof pH (6.0–10.0 pH units), temperature (35–55C), and different ion con-centration on the stability of enzyme has been given the special attentionfor study. Potential of individual CA and the enzyme consortia (solution ofall three CAs in equal proportions) was used to evaluate their sequestrationefficiency as a function of change in Ca2+ ion concentration prior to andafter the precipitation of CaCO3.

Among three isolated CAs from bacteria, CA from P. fragi showed thehighest specific activity of 70.6 U/mg protein but was slightly less active thanBCA (74.6 U/mg protein). Among all the CAs, CA from M. luteus 2 was foundto be highly stable followed by CAs from P. fragi, M. lylae, and BCA under theobserved optimum conditions of pH ranging from 8.0 to 9.0 and temperaturerange of 35–45C. All the four CAs were found to retain 75% of their initialactivity under the influence of 0.2 M concentration of Ca2+, Mg2+, Na+, andK+. However, 0.2 M concentration of Cl− ions inhibited about 50% activityof MTCA and BCA. F−, I−, and Br− at a concentration of 50 mM potentiallyinhibited the enzyme activity of all the four CAs whereas metal ions suchas Cd2+, Zn2+, Co2+, and Fe2+ were found to increase the CA activities overthe range of 6–38%. PCA and MLCA were found to tolerate 100 mM sulfateion and 50 mM nitrate ion concentration with loss of less than 20% and 10%activity as compared with BCA and MTCA, which showed the approximate

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1430 A. Y. Shekh et al.

loss of 35% and 40% of its residual activity. MLCA retained its 70% activityat 5 mM concentration of Pb2+, Hg2+, and As3+. PCA was found to retainapproximately 70% activity at 5 mM concentration of selenium whereas theloss of 37% and 35% residual activity was observed in case of MTCA andBCA.

As compared with BCA, the enzyme consortium was found to be betterat enhancing the CO2 sequestration. Higher efficiency of CO2 sequestration(61%) was exhibited by CA consortia as compared with BCA (17.8%) underoptimum conditions (Sharma and Bhattacharya, 2010).

4. CARBONIC ANHYDRASE: IMMOBILIZATION

The question of economic feasibility arises when the enzyme is consideredfor CO2 sequestration, as it is an expensive biocatalyst. To make the processeconomically feasible, various attempts have been made to immobilize CAenzyme on or in the different materials so as to reuse it efficiently to reducethe final cost of sequestration process. The ranges of materials were testedfor enzyme immobilization by different group of scientists and are discussedbelow.

Crumbliss et al. (1988) used silica beads and graphite rods for the BCAimmobilization taking into account the rigidity as well as the inorganic andnontoxic nature of these materials. These materials were also noted to pro-vide high surface area for enzyme immobilization and are found to be suit-able as packing material into a column for flow bioreactor application.

The immobilization of BCA on graphite rods occurs through the amidelinkage between primary amines from lysyl residues of enzyme and the acti-vated N-hydroxysuccinimide ester surface of the graphite rod. The activationof graphite rod for surface carboxylate groups occurs through the oxidationof it under radio frequency (rf) O2 plasma reactor for 10 min at 100 mTorrO2 pressure and of 50 W rf power. Then, the reaction of the graphite rodswith N-hydroxysuccinimide in presence of carbodimide-coupling reagent inp-dioxane for 4 hr with stirring under N2 leads to the formation of active esterintermediate. Following this, the graphite rods are rinsed with dry dioxane.The activated rods were then placed in 2 ml BCA solution (1.8 × 10−4 M,pH 8, room temperature) with continuous stirring for immobilization of BCA.Unreacted ester groups were masked by addition of solid glycine. After 90min, rods were taken out, rinsed with Tris buffer (0.1M, pH 8), and storedin buffer at 4C.

For the immobilization of BCA on gel beads, the calcium pectinate gel(CPG) beads were initially slurried with glutaraldehyde solution (10 ml, 2.5%)for 1 hr and then glutaraldehyde solution was replaced by phosphate buffer(pH 8) with BCA of strength 3.3 × 10−4 M and slurried again for 1 hr. Fi-nally, bead-enzyme complex (BCA-CPG) was rinsed and stored in Tris buffer

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Carbonic Anhydrase for CO2 Sequestration 1431

(0.1 m, pH 8) at 4C. The surface covered through immobilization of BCA onsilica beads and graphite was found to be 1.5 × 10−5 m mol/m2 and 1.7 ×10−3 m mol/m2, respectively.

BCA was assessed for esterase activity with p-NPA as a substrate andkinetic parameters have been evaluated. K′ENZ (apparent second-order rateconstant) for BCA on graphite rods and CPG beads was found to be 5.6 × 102

M−1s−1 and 2.6 × 102 M−1s−1, respectively, at 25C in Tris buffer (0.1 M, pH8).Under the similar experimental conditions KENZ was found to be 9.1 × 102

M−1s−1. pKa values for immobilized CA on graphite rods, CPG beads, andfree enzyme were calculated as 7.41, 6.75, and 7.0 respectively. Upon thestorage for 50 days under similar conditions as mentioned previously, greaterthan 97% enzyme activity was observed for CPG-supported immobilizedenzymes and 85% for enzymes immobilized on graphite rods. In the case ofBCA-CPG beads, loss of 15% activity was noted after 500 days of storage.Operational stability of enzyme immobilized on BCA-CPG beads has alsobeen tested at various temperatures in two different experimental conditionsand are discussed in Table 1 (Crumbliss et al., 1988).

Bond et al. (2001) immobilized the standard BCA in chitosan alginatebeads, which were synthesized from 20% w/v alginic acid and then added to0.2 M CaCl2 solution with 2% w/v chitosan. Optimization of immobilizationmatrix material to support less protein leakage has been done. The cross-linking solution was under continuous stirring. A total of 3 ml of sample wastaken out for enzyme quantification after 1 hr of stirring. Forty beads weresynthesized per milliliter of alginate solution. After preparation, beads weretaken out, washed twice with distilled water, and 3 ml of washing solutionwas taken for quantitative estimation of enzyme. Complete evaluation oftotal and individual losses of protein during preparation and storage (37C,110 rpm) has been carried out. Amount of nitrogen present in the beads wasalso determined, which was found to increase with increase in molecularweight of chitosan. Beads prepared at pH 5.0 with medium molecular weightchitosan alginate were more suitable for BCA immobilization. Best overallretention results were obtained at pH 2.0, where the total loss of enzyme(during preparation and storage) was only 9.104%; however, beads prepared

TABLE 1. Operational stability of enzyme immobilized on BCA-CPG beads at varioustemperatures

Operational Time period for retention Time period for retentiontemperature of 50% activity of 50% activity(C). (flow reactor) (batch reactor)

23 15 days 30 days40 24 hr 60 hr60 30 min 60 min

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1432 A. Y. Shekh et al.

TABLE 2. Kinetic parameters of CA immobilized on alkyl-substituted sepharose 4B material

Km (app) (mM) Kcat/Km (app)

Octyl-sepharose 0.53± 0.05 80± 2Dodecyl-sepharose 0.31± 0.03 50± 2Palmityl-Sepharose 0.21± 0.03 31± 1

at pH 5.0 are more preferable because of high enzyme stability, though theenzyme loss is 28.167% (Bond et al., 2001; Simsek-Ege et al., 2002a, 2002b).

Hosseinkhani et al. (2003) carried out an adsorptive immobilization ofpartially unfolded CA on hydrophobic adsorbents. This concept of immobi-lization of partially unfolded CA was evolved because native CA does notinteract with hydrophobic adsorbents. Therefore, heat-denatured CA was ad-sorptively immobilized on alkyl-substituted sepharose 4B (octyle-, dodecyl-,and palmityl-substituted sepharose 4B) (Azari and Gorgani, 1999). It wassuggested that the interaction of relatively long alkyl chains such as palmitylgroups present on the surface of sepharose may ensure sufficient hydropho-bic interaction to occur, leading to the irreversible process of adsorption(Hosseinkhani et al., 2003).

Specific activities for CAs immobilized on different materials were de-termined and are summarized in Table 2.

Bhattacharya et al. (2003) immobilized CA on 35–45 mesh particles ofiron filings collected from a lathe machine. They treated this inorganic sup-port material with organofunctional silane (γ –aminopropyltriethoxysilane)for silanization. It has been stated that silane and the available oxide groupson carriers reacts with each other, resulting in an organic functional groupswith which the coupling of enzymes can occur. In another attempt, a thinlayer of glass which was primarily coated on iron filings of 40-60 mesh andwas subsequently used for direct immobilization of CA through cynogenbromide (CNBr)–mediated coupling. In addition, the CA has been immobi-lized by copolymerization in methacrylic acid polymer beads, resulting fromthe reaction of methacrylic acid with methacrylic m-fluoroanilide nitrate. Onaverage, about 50 mg of CA/g of matrix (incubated in Hepes buffer, pH8.0, containing 1 mg/ml BCA) was used for immobilization. These methodsshowed excellent efficiency for immobilization, which was found to be over85% with 98% retention activity. Leakage of the protein from immobilizedmaterial is always the matter of concern and it is stated that the efficiencyand activity of the immobilized enzyme may get affected by leaching.

Enzyme leaching was found to be higher with a methacrylatecopolymer–coupled enzyme as compared with other immobilized en-zymes, whereas dicarboxycarbodi-imide (DCC) and carboxy-coupled en-zymes showed the least leaching even after 30 cycles of use. Moreover,total loss of enzyme activity due to leaching has not been determined. The

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Carbonic Anhydrase for CO2 Sequestration 1433

loss of catalytic activity was attributed to the structural modification of theprotein. Studies on pH and thermal stability have also been carried out andthe optimal pH for soluble CA was found to be in the range of 8.5–9.0,whereas for the immobilized CA it was in the range of 9.5–10.0 pH units.Thermal stability of both kinds of enzymes were also determined in whichit was found that the soluble enzyme was more susceptible for deactivationas compared with the immobilized enzyme. At 65C, the activity of solubleenzyme decreased drastically as compared with the activity of immobilizedenzyme (Bhattacharya et al., 2003).

Ozdemir (2009) immobilized the standard BCA within polyurethane(PU) foam at room temperature. PU foam was found to be a good sup-port material for the CA immobilization because of its high hydrophilic andporous polymeric characteristics. Foam was synthesized after polymerizationand incubated for at least 2 hr prior to use. The enzyme immobilized withinPU foam was found to retain its 100% activity at room temperature over thetested period of 45 days, which was the limiting time period for the freeenzyme to retain its activity even when stored at 40C. Kinetic parameterswere also studied and reported, in which Michaelis-Menten constant (Km)and Kcat values for free BCA were calculated as 12.2 mM and 2.02 s−1 (forp-NPA as a substrate in tris buffer, with presence of 10% acetonitrile). Theratio of Kcat to Km (Kcat/Km) was reported as 166.4 M−1s−1. The Km valuefor immobilized CA was found to be 9.6 mM. However, the values for Kcatand Kcat/Km could not be estimated for the immobilized CA because afterimmobilization the enzyme and foam became composite material and hencethe quantification of enzyme within the PU foam could not be done. Beyondthe activity comparison, an immobilized enzyme within PU foam was foundto be more stable and reusable (up to 7 cycles) at an optimum temperaturerange of 35–45C while the biocatalyst lost its activity at 60C (Ozdemir,2009).

Prabhu et al. (2009) from synthesized and tested different biopolymer-based materials for immobilization of CA-enriched microbial cells as well aspartially purified CA. The tools such as FTIR, SEM, and XRD were usedfor characterization of materials. However, all materials showed reason-ably good affinity for immobilization; chitosan-NH4OH beads, multilayeredbeads, and alginate beads were shortlisted, as they had better affinity forenzyme as well as for whole-cell immobilization. After whole-cell immo-bilization, the esterase activities, of chitosan-NH4OH beads, multilayeredbeads, and sodium alginate using p-NPA as a substrate, were found tobe 42 (which was highest activity among all the material tested), 36, and30.5 U/ml, respectively, as compared with 27.15 U/ml for the free organism.Highest esterase activity for microbial cells immobilized on chitosan-NH4OHbeads were attributed toward the presence of hydroxyl group on chitosan-NH4OH beads, which facilitates the adsorption of microbial cells on thematerial.

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Zhang et al. (2009) immobilized the enzyme in poly(acrylic acid-co-acrylamide)/hydrotalcite (PAA-AAm/HT) nanocomposite hydrogels. Thesenanocomposite hydrogels were used as such or in activated form for immo-bilization. First and foremost, hydrotalcite was prepared and then modifiedby intercalating with SMAS. Then, organically modified HT was used for insitu polymerization that leads to PAA-AAm/HT nanocomposite hydrogels.Later on, this material was subsequently activated by N-hydroxysuccinimidein presence of DCC at 50C, leading to the activated hydrogels, which wasthen used for the immobilization carried out at 4C for 4 hr. The immobi-lization capacity of nonactivated (because it is used as such) nanocompositehydrogels as well as activated hydrogels increased with increasing contentof the SMAS-HT because it increases the water-absorbing capacity of hydro-gels. Activated hydrogels were greater, almost twofold, than nonactivatedhydrogels (Zhang et al., 2009).

5. BIOREACTORS FOR CO2 SEQUESTRATION

Initial studies on CO2 sequestration with free as well as immobilized CA ledthe foundation for employing the entire process to the bioreactor with theultimate aim of taking the technology to the industry so that abatement isimplemented at source in the best possible manner.

Because of cost, maintenance, safety, and environmental issues relatedto the use of alkalomine-based CO2 separation systems, alternative ap-proaches of using CA as a catalyst into the reactor system were found tobe of great promise (Cowan et al., 2003).

Cowan et al. (2003) tested a CA membrane reactor that included asandwiched phosphate buffer containing CA between two polypropylenemembranes. The aqueous phase was varied in thickness in the range of70–670 µm by means of annular spacers. Liquid membrane fluid was constantthroughout the operation. Air-mixed CO2 was used as feed gas in the rangeof 0.04–1.0% (pCO2 ranging from 40.52 to 1013.00 Pa) at 1 atm. Argonwas used as a sweep gas. Polysulfone humidifiers in reverse were used tohumidify both kinds of gases. All the gas flows were monitored by flowcontroller. Permeate gases (N2, O2, Argon, H2O, and CO2) were analyzed bymass spectrophotometer. Based on flow rate and composition of feed andsweep gases, the separation performance of the reactor was calculated.

Performance of the reactor was tested for various parameters (varying inrange) such as CA concentration, buffer concentration, and thickness of theliquid medium; pH of medium; CO2 concentration; temperature; and humid-ity (Table 3). Under the optimum conditions of parameters tested, separationperformance of the reactor in correspondence to CO2 permeance was foundto be 4.71 × 10−8 mol m−2 Pa−1 s−1, whereas selectivities of CO2 versus N2

and O2 were found to be 1,090:1 and 790:1, respectively. Optimizations of

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Carbonic Anhydrase for CO2 Sequestration 1435

TABLE 3. Parameters tested for the performance of CA membrane bioreactor

CA Buffer Thickness of CO2concentration concentration the liquid concentration Temperature Humidity(mg/ml) (mM) medium (um) pH (%) (C) (%)

1, 2, 3, 5. 20, 50, 75,100.

70, 180, 330,490, 670

6.80, 7.04, 7.5,8.0, 8.5.

0.04–1.0 20, 25, 30, 37,40.

4, 27, 53,74.

all the individual parameters were interdependent and optimized as a func-tion of one or the other parameter. Further process of improvement is underpipeline for understanding the reactor design and increasing the separationefficiency of the CA membrane reactor.

A novel spray reactor with immobilized CA as a catalyst was used forthe very first time with the aim of capturing and solubilization of CO2 fromemission streams by allowing catalytic contact with spraying water. The en-zyme was immobilized on silica-coated steel matrix by coupling method(Bhattacharya et al., 2003). Spraying water rather than steady solution phasewas studied to speed up the solubility of CO2. The bioreactor was operatedunder different conditions of exhaust/emission gas stream (concentration aswell as flow) water flow. The effects of immobilized matrix pore size andenzyme load on CO2 reduction have also been studied. The optimal CO2

reduction was obtained with the reactor design that allows the horizontal in-flow and outflow of the emission gas (temperature 60C) and water sprayedfrom vertical position. Performance of bioreactor was evaluated by keepingCO2 concentration of emission gas as constant (in the range of 33–40%) andvarying the flow rate and vice versa by keeping the emission gas flow rateconstant (at 4.5 L/min) and varying the CO2 concentration of emission gaswith constant water flow of 2.5 ml/min for both cases. The reduction of CO2

was found to be optimum with the emission CO2 gas concentration of 70%and flow rate of 5–7 L/min with porous matrix size of 2 µm having enzymeload of 2 mg/ml in the reactor when water was sprayed at the optimal flowof 8 ml/min. All these optimal values have been taken from the plateauphase beyond which considerable decrease in the CO2 reduction capacity ofbioreactor has been observed. However, CO2 reduction capability has alsobeen studied comparatively for single and multiple reactor system where theconcept of multiple reactors was found to be more efficient for extractionof CO2 emission gas stream. It has also been suggested that an alkalinepyrogallol spray column becomes necessary when the oxygen and sulfurcontents are high in some gas emissions (Bhattacharya et al., 2003). Follow-ing these scientific findings of Bhattacharya et al. (2003), no new findingsrelated to CA-driven CO2 sequestration by means of bioreactor approachwere published until 2009.

Recently, in early 2010, development of a hollow-fiber membrane reac-tor for removal of low concentration CO2 gas from mixed gas stream was

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reported. In the hollow fiber membranes, two bundles of polyvinylidenefluoride (PVDF) were arranged in a parallel manner in a tube module. A CA-immobilized lab-made material—PAA-AAm/HT—was used to fill the spacebetween fibers. CO2 at low concentration has been effectively separatedfrom mixed gas streams by this hollow-fiber membrane reactor. Separationperformance of the reactor has been investigated in detail with respect tothe effect of the concentration of CO2, CA, and buffer. The effect of flowrate of sweep gas (Vs), feed gas (Vf), and operational temperature has alsobeen evaluated. Optimal operating conditions for reactor were evaluated andincludes the inlet CO2 concentration of 0.1% (v/v), Tris–HCl buffer of pH 8.0(20 mM), temperature 20C, VS (300 mL/min), VF (100 mL/min), and 1g/LCA. At these best operating conditions, separation selectivity of CO2 over N2

and of CO2 over O2 was found to be 820 and 330, respectively, and CO2

permeance was noted as 1.65 × 10−8 mol/m2 s Pa. Stable separation per-formance of the reactor was noted even for extended runs of 30 hr (Zhanget al., 2010).

6. CONCLUSIONS AND FUTURE ENDEAVOURS

Unveiling of new classes of CAs in various microbes undoubtedly indicatesthe crucial role of this enzyme in prokaryotic systems and, in the future,discoveries of new classes of this enzyme should not be taken as surprise.However, studies regarding the structure, enzymology, and regulation of CAmay completely reveal its novel functions in addition to the known rolein the reversible hydration of CO2. The use of CA for CO2 sequestrationinto environmentally safe mineral carbonates such as CaCO3 is an excellentapproach (Favre et al., 2009; Mirjafari et al., 2007; Ramanan et al., 2009a).However, this process highly demands cost-effectiveness of the system. Useof CA for enhancing the hydration of CO2 tends to decrease pH as a result ofrapid generation of protons into the reaction medium. Drop in pH of reac-tion medium into acidic range may greatly hinder the precipitation of solidcarbonates (Favre et al., 2009). Keeping in mind the large-scale applicationof CA-driven CO2 sequestration into mineral carbonates, the development ofan adequate and economical means of pH control is presently a major re-search need. With the available research on the CA-driven processes for CO2

sequestration, the smart efforts are needed with respect to choosing the rightmaterial and right technique for immobilization of CA so that the enzymeleakage problem can be conquered and the reusability can be enhancedwhich eventually contributes toward the improved economic feasibility. Thewhole bacterial cell (with extracellular CA production) in a free as well asan immobilized state can strongly overcome, to some extent, the economicaland physiological issues related to use of CA for CO2 sequestration purpose.The approaches that deal with the direct sparging of CO2 gas in presence of

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CA/whole cell in free/immobilized form into hydration/sequestration mod-ule of the reactor will be more practical and easy to apply at the site ofemission. Moreover, the simulation of available research findings along withsome new approaches can collectively make the CA-driven CO2 sequestra-tion processes a successful venture and can shed more light on this issue innear future.

ACKNOWLEDGMENTS

The authors are thankful to all the research fellow colleagues who helpedto improve this article considerably. The authors also thank Department ofBiotechnology, New Delhi, and Department of Science and Technology,New Delhi, for financial support.

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