Journal Pre-proof

Bioprocess development for efficient conversion of CO2 into calcium

carbonate using keratin microparticles immobilized Corynebacterium
flavescens

Tanvi Sharma, Ashok Kumar

PII: S1359-5113(20)30231-2
DOI: https://doi.org/10.1016/j.procbio.2020.10.009
Reference: PRBI 12182

To appear in: Process Biochemistry

Received Date: 12 February 2020

Revised Date: 10 September 2020
Accepted Date: 14 October 2020

Please cite this article as: { doi: https://doi.org/

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Bioprocess development for efficient conversion of CO2 into calcium carbonate using
keratin microparticles immobilized Corynebacterium flavescens

Tanvi Sharmaa, and Ashok Kumara*

a
Department of Biotechnology and Bioinformatics, Jaypee University of Information
Technology, Waknaghat, Solan, India
Corresponding author: [AK] ashok.nadda09@gmail.com

Graphical abstract

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Highlights
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 A novel strain Corynebacterium flavescens has been isolated from cow saliva.
 Effect of C. flavescens on the precipitation of CaCO3 was studied under
optimized conditions.
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 C. flavescens cells were immobilized onto highly porous keratin

microparticles.
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 Covalently immobilized C. flavescens cells showed increased CaCO3

production as compared to its free counterpart.

Abstract
Conversion of CO2 into calcium carbonate using microorganism is one of the promising
methods of reducing the increased level of CO2 in the atmosphere. In the present study, a

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novel strain of Corynebacterium flavescens was isolated from cow saliva and screened for
CO2 hydration activity. First of all, the effect of C. flavescens on CaCO3 precipitation was
studied under optimized conditions. Various factors such as CO2 concentration, protein
concentration, pH of the reaction system, and temperature were then optimized for maximum
CO2 precipitation into CaCO3 using free cell lysate. After that, C. flavescens cells were
immobilized onto highly porous keratin microparticles functionalized with glutaraldehyde
(0.6 % v/v) to enhance the CO2 conversion efficiency. Maximum immobilization of bacterial
cells was achieved after 25 h at 4oC under shaking conditions. The covalently immobilized C.
flavescens particles showed a 1.35-fold increase in CaCO3 production compared to their free
counterparts. Under repeated batch conditions, the relative production of CaCO3 for

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immobilized and free cells was 53.46 and 22.15%, respectively, after 10 cycles. This study
demonstrated the advantages of keratin microparticles as a support for the immobilization of

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C. flavescens and the efficient conversion of CO2 to CaCO3.

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Keywords: C. flavescens, CO2, keratin particles, immobilization, CaCO3
1. Introduction
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Carbon dioxide (CO2) is a major greenhouse gas (GHG) which plays a key role in
global warming. In addition, various government organizations recommend the use of
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alternative energy sources with the least carbon emissions to protect nature from the harmful
effects of GHGs. It is therefore necessary to prevent the excessive emission of CO2 into the
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environment [1-3]. Various methods have already been reported, such as the use of materials,
chemicals, and microbial enzymes for the conversion of CO2. Among them, the enzymatic
conversion of CO2 is considered to be one of the most promising strategies due to the eco-
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friendly nature of the enzyme and the lower bioprocess cost. Some algae, bacteria, and
cyanobacteria use their carboxylating enzyme and play an important role in alleviating
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increasing levels of CO2 [4, 5]. Many bacterial species with carbonic anhydrase (CA) such as
Aeribacillus pallidus, Bacillus schlegelii, Neiserria gonorrhoeae, and Bacillus subtilis have
been investigated for CO2 conversion [6]. CA contains metal ion at its active site and
converts CO2 to bicarbonate, which can be further converted to CaCO3 in the presence of
calcium ion according to the following equation:
CO2 + H2 O → HCO−
3 +H
+

Ca2+ + HCO−
3 → CaCO3 + H
+

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If atmospheric CO2 can be fixed into CaCO3, then we have an environmentally friendly
product that can also be used as raw material for various industrial applications, such as
cement, ceramics, sugar refining, glass, iron and steel production units [7]. CA can catalyze
various hydrolytic reactions, including hydration of carboxylic acid, halides, urea and
hydrolyzable substrates [8]. This enzyme is either extracellular or inside (intracellular) the
cytoplasm of the bacteria [9]. The catalytic efficiency of CA is 108 M−1s−1 and catalyzes the
reaction very quickly.
Despite the high rate of catalysis, its industrial applications are limited due to its low
stability and poor reusability [10]. The major disadvantages of using whole-cell as a

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biocatalyst are the occurrence of metabolic by-products and membrane can act as a mass
transport barrier [11]. Over the last decade, these problems have been overcome by

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immobilizing the enzymes and microbes having CO2 conversion efficacies onto various
supports such as chitosan, alginate, polyurethane foam, and magnetic nanoparticles [12-15].
Previous studies have suggested that whole-cell microorganisms have shown a significant
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increase in CaCO3 production by various microbial species, such as Geobacillus
thermoglucosidasius, Cyanobacteria, and, Cholerlla [16-18]. Moreover, the immobilization
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of whole cells could reduce the time-consuming and costly steps required for the purification
of enzymes [19]. Generally, purified enzymes are preferred for immobilization and
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biocatalytic transformation. Specifically, the immobilization of the whole cell was preferred
in the case of an enzyme with low stability and short half-life. Whole-cell immobilization, on
the other hand, gives microbes stability against environmental stress such as pH, salts,
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solvents, self-destruction and protects the cells from shear forces. The presence of cellular
envelope helps in stabilizing the cells and enables its application in environmental stress
conditions. Furthermore, the viability, activity and productivity of immobilized cells can be
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maintained for a long time, resulting in improved operational stability [20]. However, the
conventional supports used for immobilization exhibit non-uniformity and require more time
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for material preparation, which reduces the immobilization efficiency. New support with high
porosity and stable structure must therefore be used. Keratin particles extracted from chicken
feathers act as support for the immobilization of whole cells. These highly porous keratin
particles can be considered cost-effective and biocompatible.
In this study, the feasibility of using C. flavescens for the precipitation of CO2 into
CaCO3 was studied. In addition, bacterial cells were immobilized onto keratin particles to
enhance calcium carbonate production. The results showed that immobilized C. flavescens

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cells onto keratin particles had a significant increase in CaCO3 production compared to free
cells. Here, we demonstrate that keratin particles immobilized cells are a green biocatalyst for
the conversion of CO2 into calcium carbonates.

2. Methods
2.1 Materials
Various buffer components, nutrient broth, calcium chloride, bovine serum albumin
(BSA), Bradford, calcium carbonate,and glutaraldehyde were obtained from Himedia,
Mumbai, India. All the chemicals used were of analytical grade and were obtained from
standard sources.

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2.2 Isolation and identification of bacterial strain
The sample was collected from six years old cow saliva from District Mandi,

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Himachal Pradesh, India, and identified by 16S rRNA sequencing based on nucleotide
homology and phylogenetic analysis by Bioreserve Biotechnologies Pvt. Ltd., Hyderabad,
India [21].
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2.3 Organism and growth condition

The C. flavescens was cultured in the nutrient broth and incubated at 37°C and 120
rpm for 24 h. Subsequently, when the optical density of culture reached 0.6-0.7 at 600 nm the
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cells were harvested by centrifugation at 5000 g for 20 min at 4°C. The cell pellet was
washed twice with 50 mM sodium phosphate buffer (pH 7.5) and stored at 4°C until further
use.
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2.3 Carbonic anhydrase assay

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The electrometric method (Wilbur-Anderson) for CA activity was followed with

minor modifications [22]. The CO2 saturated water was prepared by bubbling CO2 gas into
the deionized water at a pressure of 1.4 bar. To initiate the reaction, a reaction mixture
containing 3 mL of Tris HCL (pH 8.0), 0.1 ml of cell lysate, and 2 mL of CO2 saturated water
was added. CA activity was calculated in Wilbur-Anderson units per mg of protein. The
protein content was estimated using the Bradford method [23].

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2.4 Conversion of CO2 into calcium carbonate
The potential of C. flavescens for CO2 conversion was determined as described by
[24] with minor modifications. The precipitation of CO2 into CaCO3 was carried out in a 50
mL total reaction mixture containing 2.0 mL Tris buffer (1M, pH 8.0), 23 mL of 2% (w/v)
calcium chloride solution, 23 mL of CO2 saturated water and 2.0 mL (0.05 mg/mL) of cell
lysate in phosphate buffer (50 mM, pH 7.0). The reaction was performed for 10 min at room
temperature.
The precipitate formed after 10 min was recovered by centrifugation. The sediment
of precipitates was lyophilized overnight in order to obtain a dry powder [25]. CaCO3
precipitates were then weighed to determine the amount of carbonate synthesized during the

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enzymatic reaction. Various parameters, such as the effect of CO2 concentration, protein
concentration, pH, and temperature were studied to investigate the performance of CO2

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conversion with C. flavescens.

2.5 Synthesis of porous keratin microparticles

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The chicken feathers were collected from a chicken processing plant near Solan, Himachal
Pradesh, India. The chicken feathers were then hydrolyzed under alkaline conditions using
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chemical treatment as previously reported [26]. The protein hydrolysate was freeze-dried and
the keratin powder was collected. The synthesized keratin particles were washed three times
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with double distilled water. The surface morphology of keratin particles was studied using
scanning electron microscopy (SEM).
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2.6 Whole-cell immobilization

For covalent immobilization, the keratin particles (1 g) were functionalized with 0.2
% (v/v) glutaraldehyde for 2 h at 25°C as previously reported [27]. This protocol allows the
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introduction of two molecules of glutaraldehyde per primary amino group in the support [28].
After functional activation, keratin particles were washed twice with phosphate buffer to
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remove excess glutaraldehyde. C. flavescens were immobilized at a cell concentration of 0.5

g DCM g−1 of support and incubated for 30 h at 4°C. The immobilization yield was
calculated as previously described [29].
The keratin particles immobilized cells of C. flavescens were also characterized for
CO2 conversion efficiency at different glutaraldehyde concentrations (0.2-1.4%), incubation
time (5-35 h) and temperatures (4-55°C). In addition, the conversion of CO2 by free and

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immobilized cells was performed for ten cycles, after each cycle cells were recovered by
centrifugation and used for the next cycle.

2.7 Instrumental analysis of CaCO3

The CaCO3 precipitates were lyophilized to obtain dry powder of CaCO3. SEM was
performed with JSM7401F (JEOL) to determine crystal morphology. The Fourier transform
infrared (FTIR) spectra (model Varian 7000 FTIR) was used to determine the chemical bonds
and functional groups present in the precipitates. The CaCO3 precipitates were scanned in the
range of 500-2500 cm-1. Compositions of the precipitated solid crystals were examined using
X-ray diffraction (XRD). Calcium carbonate obtained from Himedia was used as a control.

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3. Results and discussion

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3.1 Identification of bacterial strain
The DNA was isolated from the bacterial strain and the DNA analysis was performed
by agarose gel electrophoresis and then the dendrogram was made using the tree joining
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method. The bacterial isolate was identified as Corynebacterium flavescens using 16 rRNA
sequencing and the accession number of the organism was MN982752 deposited in NCBI
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[30].
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3.2 Conversion of carbon dioxide into calcium carbonate using cell lysate
The CO2 conversion efficacy of cell lysate was studied by calculating the amount of CaCO3
synthesized. The cell lysate having 144 U/mg of enzyme resulted in the formation of 65.12
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mg CaCO3 at 30oC. The CO2 conversion efficiency of the enzyme produced from C.
flavescens was much higher compared to CA from B. pumilis (33.06 mg CaCO3/mg of
protein) [31]. In addition, various factors affecting CO2 conversion efficiency were studied.
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The results indicated that a change in CO2 concentration could have an impact on the CO2
conversion efficiency. The CO2 conversion efficiency decreased from 82.35% to73.63% with
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an increase in the CO2 concentration (Fig. 1a). The maximum yield of CaCO3 observed with
2g/L CO2 was 66.11 mg. A further increase in CO2 concentration did not affect the
precipitation of CO2 into CaCO3. This is due to the fact that CO2 absorption was necessary to
overcome the increased mass transfer resistance from the gas phase and liquid phase. In short,
a higher concentration of CO2 required a higher amount of immobilized cells or a larger
surface area for a biocatalytic reaction. Thus, CO2 absorption encountered more resistance
with a lower ratio of cells resulting in a decrease in CO2 conversion [32]. As shown in

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(Fig.1b), the effect of protein concentration (0.1-13mg/mL) on the CO2 conversion efficiency
was studied; with an increase in protein concentration, relative CaCO3 (%) first increased and
then decreased. The optimum protein concentration was found to be 0.05 mg/mL. At lower
protein concentration, limited number of cells combined with the CO2. However, a further
increase in protein concentration reduced the conversion rate of the CO2.
Most precipitation occurs under alkaline conditions, at pH below 8.3 the bicarbonate
ions are converted to carbonate and there will be no reaction. Under alkaline conditions,
bicarbonate ions exist in equilibrium with carbonate ions and in the presence of calcium ions,
calcium carbonate precipitates are formed [33-35]. The higher concentration of hydroxide
ions is required for the absorption of CO2 into the reaction mixture. Maximum CaCO3 (70.69

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mg) was therefore formed at pH 8.5 following a decrease in CaCO3 formation (Fig. 1c).
Ramanan et al., [36] reported the formation of calcium carbonate using tris buffer at pH 8.3.

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The formation of CaCO3 using tris buffer at pH 8.4, 9.4, 10.5 was also studied by Favre et al.,
[37]. Moreover, the whole-cell catalyst was more stable at pH 7.0 than the high-pH
condition, which could be the reason for maximum precipitation at pH 8.5 [38]. The cells
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were no longer active at high alkaline pH which might be attributed to a decrease in CaCO3
formation. In addition, the effect of temperature on CaCO3 formation was studied and
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maximum CaCO3 formation occurred at 40oC (Fig. 1d). There was decrease in the amount of
CaCO3 with an increase in temperature, since the solubility of CO2 in water decreases with
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increase in temperature [39]. These results are similar to that of bovine carbonic anhydrase
(BCA) studied by [40]. Previously, three indigenous CAs (M. lyale, P. fragi, M. leutus)
reported better CO2 conversion at 45oC compared to BCA [41]. Therefore, C. flavescens not
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only enhanced the CO2 hydration reaction but also promoted the CaCO3 formation.

3.3 Bioprocess development using whole-cell immobilized microreactors

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During the biotransformation process, the immobilization of whole cells is crucial for
improving the stability of the free cells. A proper immobilization may increase cells stability
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by rigidifying the cell structure and by partitioning deleterious compounds from the micro-
environment [42]. Thus, C. flavescens were covalently immobilized onto keratin
microparticles to improve the CO2 conversion efficiency. The morphology of synthesized
keratin particles is shown in (Fig. Sa). From the SEM images, keratin particles were tightly
packed with small pores. The pore dimension is sufficient to immobilize the C. flavescens
cells. The particles were functionalized with glutaraldehyde and incubated with cells to
immobilize C. flavescens onto keratin particles. The morphology of whole-cell immobilized

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on keratin particles as shown in (Fig. Sb) revealed that whole-cell was bound on the surface
and inside the pores in the keratin particle. Moreover, the effect of different glutaraldehyde
concentrations on immobilization was studied within a range of 0.2-1.4% (v/v). Mostly, in
the lipid bilayer of cell membrane glutaraldehyde reacts with the protein and covalently binds
cells [43]. The optimum glutaraldehyde concentration needed to achieve the highest cell
immobilization (CI) yield and relative efficiency of CaCO3 production was found to be 0.6%
(v/v) (Fig. 2a). Using higher glutaraldehyde concentrations, there were multiple cross-linking
points on the keratin particles that caused spatial hindrance among cell aggregates, leading to
lower cell immobilization yield [44]. Thus 0.6% glutaraldehyde was used to activate the
support in further studies. The results obtained for the study of glutaraldehyde concentration

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effect in the functional activation of the support are different from those obtained using
graphene oxide nanosheets, where 1.0% glutaraldehyde concentration was found to be

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optimum [45]. Earlier studies showed that 0.1% (v/v) glutaraldehyde concentration was
optimum for the immobilization of Bacillus licheniformis onto alginate [46]. The optimum
temperature for immobilization of cells was 4oC with maximum cell immobilization yield and
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higher relative CaCO3 production efficiency (Fig. 2b). These results showed that a lower
temperature is beneficial for immobilization compared to a higher temperature. Similarly, in a
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previous study, maximum CI yield was observed at 4oC [47]. In another study, the optimum
temperature for the immobilization of esterase onto mesoporous silica was found to be 10oC
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[48].
Initially, the CI yield was improved by increasing the incubation period up to 25 h
which later became stable with a longer incubation period up to 35 h (Fig. 2c). Further
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increase in the incubation time did not affect the cell immobilization onto keratin particles.
The same trend was observed for relative CaCO3 production efficiency by immobilized cells
under similar conditions. Previously, [49] reported that the optimum incubation time for
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immobilization of M. tundra was 24 h. Finally, the CaCO3 production was evaluated by

immobilized and free cells for 10 successive cycles. The amount of calcium carbonate formed
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by the immobilized and free cells in the first cycle of reuse was 86.71 mg and 64.02 mg,
respectively. After ten cycles of recycled use for CO2 conversion, the immobilized and free
cells showed relative CaCO3 production of 53.46 and 22.15%, respectively (Fig. 2d). This is
fairly good reusability and can be attributed to the stabilization and retention of the cells in
the keratin particles. In addition, immobilized cells can be recovered from the solution more
easily and handled without difficulty. The slight decrease in CaCO3 formation after each
cycle may be due to the leaching of cells, adsorption of keratin particles to the reaction tube

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during centrifugation, and minor loss of keratin particles during the decantation of
supernatant. In a previous study, CA immobilized by precipitation-based approach in a
nanoscale enzyme reactor presented high operational stability. After ten times of reuse for
CO2 conversion, the relative CaCO3 formation was 89% [50]. CaCO3 is a valuable chemical
used in various industries such as fillers for paints, plastics, paper and food and
pharmaceutical applications [51]. Remarkably, these results suggest whole-cell
immobilization as an effective technique for higher CaCO3 production.

3.4 Instrumental analysis of CaCO3

CaCO3 exists in different crystal forms, such as vaterite (spherical), calcite (rhombic) and

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aragonite (needle-like). In the SEM analysis, vaterite was the most dominant phase and few
calcite forms were also formed in the presence of immobilized cells (Fig. 3). SEM coupled to

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EDX also made it possible to carry out elementary and punctual chemical analysis of the
material, with indicated Ca, O, C peaks in which calcium and oxygen peaks were more
prominent, followed by the carbon peak. Furthermore, various studies have shown that calcite
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is the main phase obtained upon CaCO3 precipitation in the presence of CA [51]. From the
FT-IR spectra, the test sample showed major peaks at 1465, 1420, 874, and 712 cm−1 (Fig.
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4a). Similar peaks were observed with a control sample. In XRD analysis, vaterite form was
found to be a major form of crystal with a corresponding diffraction peak at 25.46. Whereas,
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diffraction peak at 29.48 corresponded to calcite form (Fig. 4b). It was previously reported
that the vaterite and calcite phase were formed in the presence of CA [52]. This immobilized
C. flavescens cells thus offers an alluring prospective for CaCO3 production [53,54].
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4. Conclusion
Microorganisms are commonly used to produce enzymes of industrial use. In addition, these
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results also indicated that more CaCO3 was formed when different parameters such as CO2
concentration, protein concentration, pH of the reaction mixture, and temperature were
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optimized. More importantly, this is the first report to enhance CaCO3 production from CO2
by immobilized C. flavescens cells. In future studies, strain engineering, enzyme purification,
directed evolution, and rationale hybrid design can be used to develop a mutant of this
enzyme with improved catalytic activity and stability for industrial purposes.

Acknowledgment

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 The financial support from the Jaypee University of Information Technology,
Waknaghat to undertake this study is thankfully acknowledged. Further, the authors have no
conflict of interest either among themselves or with the parent institution.
Author contributions

Tanvi Sharma (Ph.D student) performed all the experiments and wrote the manuscript.

Ashok Kumar (Assistant Professor) designed, supervised the experiments and revised the

manuscript.

Conflict of interest

The authors have no conflict of interest either among themselves or with the parent institution.

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Figures caption

Fig. 1. (a) Effect of CO2 concentration on the precipitation reaction was determined with
maximum yield at 2g/L of CO2 as 100% at temperature 30⁰ C, pH 8.0 and protein
concentration 0.05 mg/mL (b) Effect of protein concentration on precipitation reaction was
analyzed with maximum yield at 0.05 mg/mL as 100% at CO2 concentration 2g/L, pH 8.0
and temperature 30⁰ C (c) The precipitation reaction at Tris–HCl buffer (pH 8.0-10.5) was
determined with maximum yield at pH 8.5 as 100% at CO2 concentration 2g/L, protein
concentration 0.05 mg/mL, and temperature 30⁰ C (d) The optimum temperature for
precipitation reaction was analysed under standard assay conditions with maximum yield at
40⁰ C as 100% at CO2 concentration 2g/L, protein concentration 0.05 mg/mL, and pH 8.5.

Fig. 2. Immobilization of C. flavescens on keratin particles (a) Effect of glutaraldehyde

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concentration (b) Effect of temperature (c) Effect of time on cell immobilization yield and
CaCO3 production efficiency (d) Repeated batch CaCO3 production by C. flavescens free or
immobilized cell.

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Fig. 3. Morphological and elemental mapping images of CaCO3 (a) FE-SEM image of CaCO3
(b) Area selected for elemental mapping (c-f) elemental mapping images showing the
presence of d) Calcium, e) Carbon and f) Oxygen -p
Fig. 4. (a) FTIR spectra of synthesized CaCO3 using immobilized cells. The peak of control
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sample (pure CaCO3 analytical grade) was shown in figure. (b) XRD analysis of CaCO3
precipitates
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