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Biomineralization of Calcium Carbonate Polymorphs by the Bacterial Strains

Isolated from Calcareous Sites

Article in Journal of Microbiology and Biotechnology · May 2013

DOI: 10.4014/jmb.1212.11087 · Source: PubMed

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J. Microbiol. Biotechnol. (2013), 23(5), 707–714
[Link]
First published online January 27, 2013
pISSN 1017-7825 eISSN 1738-8872

Biomineralization of Calcium Carbonate Polymorphs by the Bacterial Strains

Isolated from Calcareous Sites
Dhami, Navdeep Kaur1, M. Sudhakara Reddy1*, and Abhijit Mukherjee2
1
Department of Biotechnology, and 2Department of Civil Engineering, Thapar University, Patiala 147004, India
Received: December 3, 2012 / Revised: December 29, 2012 / Accepted: December 30, 2012

Microbially induced calcium carbonate precipitation The precise role of microbes in the carbonate precipitation
(MICCP) is a naturally occurring biological process that process is still not clear. However, three hypotheses that
has various applications in remediation and restoration of seem to be responsible for the process of carbonate
a range of building materials. In the present investigation, crystallization are as follows: (i) mineralization occurs as a
five ureolytic bacterial isolates capable of inducing by-product of microbial metabolism involving either
calcium carbonate precipitation were isolated from autotrophic or heterotrophic pathways [4]. During these
calcareous soils on the basis of production of urease, passive processes, reactions, such as enzymatic hydrolysis
carbonic anhydrase, extrapolymeric substances, and biofilm. of urea or the dissimilatory reduction of nitrate and sulfate,
Bacterial isolates were identified as Bacillus megaterium, cause an increase in pH that shifts the bicarbonate-
B. cereus, B. thuringiensis, B. subtilis, and Lysinibacillus carbonate equilibrium toward the production of more
fusiformis based on 16S rRNA analysis. The calcium CO32_ and ultimately leads to the precipitation of CaCO3, if
carbonate polymorphs produced by various bacterial free Ca2+ is present, (ii) nucleation of the carbonates take
isolates were analyzed by scanning electron microscopy, place on the cell wall, either due to ion exchange through
confocal laser scanning microscopy, X ray diffraction, and the cell membrane [5] following some still poorly known
Fourier transmission infra red spectroscopy. A strain- mechanisms, or due to the support of negatively charged
specific precipitation of calcium carbonate forms was specific cell wall functional groups that adsorb divalent
observed from different bacterial isolates. Based on the cations, such as Ca2+ [24], (iii) microbial extracellular
type of polymorph precipitated, the technology of MICCP polymeric substances (EPS) seem to play an important role in
can be applied for remediation of various building materials. precipitation, either through the trapping and concentration
Key words: Calcite, vaterite, urease, Bacillus, biofilm, of calcium ions or as a result of specific proteins that
carbonic anhydrase influence precipitation [3]. Specific proteins present in
biological cellular polymeric substances cause the formation
of different CaCO3 polymorphs [15]. Bacterial surfaces
also play an important role in calcium precipitation. The
Biomineralization is the process of synthesis of inorganic presence of several negatively charged groups at neutral
mineral-like materials by living organisms. Calcium carbonate pH favors to bind to positively charged metal ions on
precipitation is a well-known example of extracellular bacterial surfaces, favoring heterogenous nucleation [2].
bacterial biomineralization, and a large number of bacterial Biomineralization of calcium carbonate results in the
genera have been observed in a range of natural environments. production of different phases of calcium carbonate as
Bacterial mineralization of calcium carbonates has found anhydrous polymorphs: calcite, aragonite, and vaterite or two
applications in the remediation (fixation) of metal- hydrated crystalline phases, monohydrocalcite (CaCO3·H2O)
contaminated soil and groundwater [33], atmospheric CO2 and ikaite (CaCO3·6H2O), but calcite and vaterite are the
sequestration [20], strengthening and consolidation of sand, most common bacterial calcium carbonate polymorphs
limestone monument repairs, reduction of permeability of [25]. Besides its scientific interest, different calcium carbonate
cement mortar, pores and cracks filling in concrete, and polymorphs have important technical implications, as in
enhancing the strength of ash bricks [7, 8]. the case of the bacterial conservation of building materials,
where the formation of coherent, durable calcium carbonate
*Corresponding author
Phone: +911752393743; Fax: +911752393738; is required [18]. The same principle applies for other purposes
E-mail: msreddy@[Link] like soil strengthening, cement and concrete protection,
708 Dhami et al.

solid phase capture of groundwater metal contaminant, as described in Achal et al. [1]. One unit of urease is defined as the
and/or the effective (long-term) CO2 sequestration via amount of enzyme hydrolyzing 1 micromole urea/min/ml. The carbonic
bacterial calcium carbonate mineralization. There is currently anhydrase assay was performed as described by Smith and Ferry [26].
great interest in the development of materials based on One unit of carbonic anhydrase activity is defined as the amount of
enzyme required to form one µmole of p-nitrophenol per minute.
biomineralization, but still very few bacteria have been
Extracellular polymeric substances (EPS) production was determined
exploited. In the present investigation, we isolated and according to the procedure described by Friedman et al. [11]. Biofilm
characterized calcifying bacterial strains from calcareous formation was established aseptically in nutrient broth containing
soils and tested their abilities to produce urease, carbonic urea-CaCl2 on glass plates (25 × 75 mm) using the crystal violet
anhydrase, extra polymeric substances, biofilm, and calcium (CV) method described by Morikawa et al. [21].
carbonate precipitates. These strains were identified based
on biochemical and molecular characteristics. The properties Precipitation of Carbonate Crystals and Analysis
of the crystals produced were analyzed morphologically All bacterial cultures were incubated at 37oC for 3 weeks in NB
and also for elemental composition. We aim to characterize media along with 2% urea and 25 mM CaCl2 (pH 8.0). The
the crystal aggregates precipitated by these bacterial isolates. precipitated carbonates were collected on Whatman No. 1 filter
The implications of these findings in understanding microbially paper by filtration, washed with sterile distilled water, and air dried
at 37oC for 48 h, weighed, and analyzed for its morphological and
induced calcium carbonate precipitation (MICCP) as well
chemical constituents.
as its applications in building materials are discussed. The precipitated carbonate crystals were analyzed using scanning
electron microscopy (SEM; ZEISS EVO 50) equipped with EDX.
For SEM analysis, the crystals were fixed overnight in 2.5%
MATERIALS AND METHODS glutaraldehyde in 0.1 M sodium phosphate buffer at 4oC, rinsed in
0.2 M phosphate buffer saline (pH 7.4) for 1 h, and dehydrated in a
Isolation and Characterization of Bacteria series of graded ethyl alcohol. The SEM observation was done
Calcareous soil samples (pH 11.0) were collected from Anantapur under the following analytical conditions: EHT = 20.00 kV, WD =
District, Andhra Pradesh, India and the bacteria were isolated by the 10-11 mm. Elemental analysis was done with an energy dispersive
serial dilution method by plating the samples on urea agar base (Hi X-ray analyzer (Bruker AXS, Quan Tax 200). In order to determine
Media, India), a urease selective medium. Five isolates, designated the morphological features of different crystals by confocal scanning
as SS3, SS5, SS13, SS15, and SS18, were selected for further laser microscopy (CSLM), the fluoresecent dye CTC (5-cyano-2,3-
studies based on their abilities to produce urease qualitatively. The ditolyl tetrazolium chloride; Polysciences Inc.) was used. Before
biochemical characterization of these isolates was done as per the the observation, the crystals were fixed for 30 min with 4%
standard protocols [13] along with morphological and physiological paraformaldehyde-phosphate-buffered saline. The slides were flooded
studies. Genomic DNA was extracted from overnight grown cultures, with freshly prepared 1.5 mM (final concentration) CTC solution.
and 16S rRNA genes were amplified as described in Karn et al. The stain solution was left to react overnight at room temperature in
[14]. The amplicons were purified with the QIA gel extraction kit the dark, and the slides were stored at 20oC for 30 min to stop the
(Qiagen, USA), and ligated into the pTZ57R/T vector as per the reaction. Before visualization with a confocal scanning laser
manufacturer’s instructions (Fermentas, USA). Ligated plasmids were microscope (LSM 510 Meta; Carl Zeiss), the slides were covered
transformed into Escherichia coli DH5α cells. Recombinant plasmids with a cover slip and fixed with DPX mountant (Hi Media). The
were sequenced using an Applied Biosystems automated DNA stain was excited at 453 nm by the use of a 590-nm long-pass filter.
sequencer (DNA Sequencing Facility, Delhi University, India). BLAST XRD spectras were obtained using an X’ Pert PRO diffractometer
analysis was performed to compare the sequences with available with a Cu anode (40 kV and 30 mA) and scanning from 3o to 60o.
DNA sequences of the NCBI. The sequences were aligned using the The components of the sample were identified by comparing them
MAFFT ([Link] program and the with standards established by the International Centre for Diffraction
alignment was manually corrected and a phylogenetic tree was Data. Fourier transform infra red (FTIR) spectra were recorded on a
constructed by the neighbor-joining method using MEGA 5.0 software Bruker Vertex 70 apparatus by the diffuse reflectance accessory
[30]. The 16S rRNA gene sequences determined in this study are technique. The spectra of the crystals were scanned in the range of
deposited in the GenBank of the NCBI under the accession numbers 400-4,000 cm-1.
KC121060 to KC121064. These cultures were deposited at Microbial All experiments were performed in triplicates. The data were
Type Culture Collection and Gene Bank, Institute of Microbial analyzed by analysis of variance (ANOVA) and the means were
Technology, Chandigarh, India. compared with a Tukey’s test. All the analyses were performed
using GraphPad Prism (5.0) software.
Enzyme Assays, EPS, and Biofilm Production
The bacterial isolates were grown in nutrient broth medium. For
urease (UA) assay, the medium was supplemented with 5 µM nickel RESULTS
chloride (NiCl2) along with 2% urea, and for carbonic anhydrase
(CA) assay, 10 µM zinc sulfate (ZnSO4) was supplemented. The
Isolation and Identification of Bacteria
final pH of the medium was adjusted to 8.0. The cultures were
incubated at 37oC in a rotating shaker at 120 rpm for 96 h. Urease
Among all the isolated bacteria, five isolates (viz., SS3,
assay was carried out as per the phenol-hypochlorite assay method SS5, SS13, SS15, and SS18) were selected based on their
 BIOMINERALIZATION OF BACTERIAL CALCITE 709

high urease, carbonic anhydrase, and carbonate precipitation. differences among all the isolates. These isolates were
Biochemical characteristics showed that these bacterial further identified by 16S rDNA sequence analysis. BLAST
isolates are Gram-positive. They are rod-shaped, and analysis revealed that SS3 had 99% similarity (100%
catalase, urease, and oxidase positive. All isolates are coverage) with Bacillus megaterium, SS5 had 99% similarity
motile except SS15, which was nonmotile. Starch, casein, (100% coverage) with B. cereus, SS15 had 99% similarity
and gelatin hydrolysis were recorded with SS3, SS13 and (99% coverage) with B. thuringiensis, SS13 had 96%
SS15, whereas SS5 and SS18 hydrolyzed casein and similarity (100% coverage) with B. subtilis, and SS18 had
esculin. Nitrate reduction was observed by SS3, SS15, and 99% similarity (97% coverage) with Lysinibacillus fusiformis.
SS18. Acid production was observed with sucrose, glucose, Phylogenetic analysis also grouped all these isolates to
arabinose, ONPG, and malonate by SS3, but only with phylum Firmicutes and family Bacillaceae (Fig. 1).
sucrose, glucose, and mannitol by SS5, SS13, SS15, and
SS18. Citrate was utilized by SS3 and SS5. SS3 and SS5 Urease and Carbonic Anhydrase Activities
isolates showed growth in a pH range of 6.5-11.5, whereas Among the isolates, Bacillus megaterium showed maximum
the other isolates had a pH range of 6-10.5. SS3 and SS18 urease activity, followed by B. thuringiensis, B. cereus, L.
were able to survive 0-8% NaCl, and the other isolates in fusiformis, and B. subtilis (Fig. 2). The maximum urease
the range of 0-7% NaCl. In the case of temperature activity observed by B. megaterium was 690 U/ml, followed
tolerance, both SS3 and SS5 were able to survive in the by B. thuringiensis (620 U/ml) on the 4th day, while B.
range of 25-50oC, SS13 at 28-45oC, SS15 at 25-45oC, cereus, L. fusiformis, and B. subtilis produced 587, 525,
and SS18 at 25-55oC. The morphological, physiological, and 515 U/ml on the 5th day, respectively. Carbonic anhydrase
and biochemical characterization results showed marked (CA) produced by B. megaterium was found to be higher

Fig. 1. Neighbor-joining tree based on bacterial 16S rRNA gene sequence data from different isolates of the current study along with
sequences available in the GenBank database.
Numerical values indicate bootstrap percentile from 1,000 replicates.
710 Dhami et al.

Fig. 2. Urease and carbonic anhydrase activities of B. megaterium Fig. 4. Calcium carbonate crystals precipitated by different
(Bm), B. cereus (Bc), B. thuringiensis (Bt), B. subtilis (Bs), and bacterial isolates after 3 weeks (values are mean ± SD).
L. fusiformis (Lf) (values are mean ± SD).

were highest over a period of three weeks at 37oC. B.

compared with the other isolates (Fig. 2). The maximum megaterium produced 187 mg/100 ml, followed by B.
CA activity was 115 U/ml by B. megaterium, followed by subtilis (178 mg/100 ml), B. thuringiensis (167 mg/100 ml),
B. cereus (90 U/ml) on the 4th day, followed by 85, 70, and B. cereus (156 mg/100 ml), and L. fusiformis (152 mg/100 ml).
60 U/ml by B. thuringiensis, B. subtilis, and L. fusiformis No crystals precipitated in the control sets.
on the 5th day, respectively. SEM studies of various crystals formed by different
bacterial isolates showed obvious differences in size and
EPS and Biofilm Production shape (Fig. 5). The size of crystals formed by B. megaterium
B. megaterium was able to produce 36.4 nmol/ml of EPS, were 30-50 µm in diameter, whereas the size varied from
followed by B. thuringiensis at 33.5 nmol/ml, L. fusiformis 15-40 µm in B. cereus, 10-50 µm in B. subtilis, 2-15 µm
at 28.6 nmol/ml, B. subtilis at 26.8 nmol/ml, and B. cereus at in B. thuringiensis, and 2-10 µm in L. fusiformis. Crystals
26.4 nmol/ml. B. megaterium also produced higher monoxenic formed by B. megaterium were spherical, oval, rhombohedral
biofilm (283 CFU/mm2) compared with B. cereus (243 as well as triangular in shape with smooth and rough
CFU/mm2), B. thuringiensis (232 CFU/mm2), L. fusiformis surfaces. The number of crystals with smooth surfaces was
(198 CFU/mm2), and B. subtilis (194 CFU/mm2) (Fig. 3). relatively more abundant than with rough surfaces. Rod-
shaped imprints of bacteria were clearly observed on the
Carbonate Crystals Analyses surface of crystals (Fig. 5b). SEM observation also showed
Carbonate crystals were precipitated by all the isolates the formation of a mucous matrix over the calcified
(Fig. 4). Carbonate crystals precipitated by B. megaterium bacterial cells (Fig. 5c). The crystals formed by B. cereus
were needle-like and layer-flake structures. The crystals
were quite irregular and approximately of square morphology.
In the case of B. subtilis, very smooth, spherical, and
ellipsoidal crystals along with embedded bacterial cells
were observed. The magnified SEM photograph of these
crystals indicated that they were packed closely and
regularly. A few bacterial cells were also seen on the
surface of crystals. The crystals formed by B. thuringiensis
were small, round, circular rings and rectangular in
morphology. In the case of L. fusiformis, the crystals had a
very rough surface with a flaky needle-like mesh formed
an around. Some slimy EPS layers were also clearly seen
in these crystals.
Confocal laser scanning microscopy (CLSM) studies
revealed strong fluorescence in the case of all crystals
Fig. 3. Extracellular polymeric substances (EPS) and biofilm
production by B. megaterium (Bm), B. cereus (Bc), B. thuringiensis (Fig. 6). Polycrystal formation was observed in the case of
(Bt), B. subtilis (Bs), and L. fusiformis (Lf) (values are mean ± B. megaterium. The crystals produced by other isolates
SD). appeared as separate and a mixture of spherical, ellipsoidal,
 BIOMINERALIZATION OF BACTERIAL CALCITE 711

Fig. 5. Scanning electron microscopic images of carbonate crystals precipitated by (a, b) B. megaterium (Bm), (c, d) B. cereus (Bc), (e,
f) B. thuringiensis (Bt), (g, h) B. subtilis (Bs), and (i, j) L. fusiformis (Lf).
712 Dhami et al.

whereas in the case of L. fusiformis, pure vaterite crystals

were observed. FT-IR spectra of all the isolates were
studied. Crystals formed by B. megaterium, B. cereus, and
B. subtilis isolates showed a strong absorption peak at
λ = 713 cm-1, which corresponds to calcite. In the case of
isolates B. thuringiensis and L. fusiformis, the absorption
peak at 750 cm-1 depicted vaterite formation.

DISCUSSION
Bacterial strains isolated from calcareous soils were able to
precipitate carbonates under the experimental conditions.
Urea agar base was used to select urease-producing
microorganisms. All the isolates produced significant
amounts of urease. Bacteria are known to hydrolyze urea
by urease for the purpose of either to increase the ambient
pH or to utilize it as a nitrogen source or energy [12]. Urease
activity increased during the first 4-5 days, but later on
decreased significantly. The carbonic anhydrase activity
was also seen to correlate with urease activity. Hydrolysis
of urea results in accumulation of both bicarbonate and
ammonia in the cell, which favors the physiological and
regulatory links between urea and bicarbonate metabolism
[28]. The incorporation of nickel into the active site of
urease is dependent on CO2/ HCO3– metabolism, which in
turn is regulated by carbonic anhydrase [22]. Calcite
precipitation is dependent on the concentration of Ca2+ and
CO32- in solution. An increase in CO32- concentration occurs
under alkaline conditions abundant in the calcium (Ca2+)
and carbonate (CO32-) ions [23]. Both urease and CA
promote this precipitation outside the cells. EPS play an
important role in the coverage of the surface by biofilms,
cell adhesion [32], and precipitation, either through trapping
and concentration or through the action of specific proteins
that influence precipitation [3]. The matrix of EPS secretions
Fig. 6. Confocal laser scanning electron microscopic images of has been described to influence calcium carbonate
bacterial carbonate crystals produced by (a) B. megaterium precipitation in a positive way [15]. Along with EPS, the
(Bm), (b) B. cereus (Bc), (c) B. thuringiensis (Bt), (d) B. subtilis
(Bs), and (e) L. fusiformis (Lf). role of biofilm in colonizing the building surface and
in reacting as a nucleation site for extracellular calcium
carbonate precipitation has been reported by Merz-Preiss
and Riding [19].
and rectangular shapes. Bacterial cells seemed to be linked SEM and CLSM analyses indicated different morphologies
to the crystals. of CaCO3 crystals induced by different bacteria. There
EDX analysis of all the isolates showed a large amount were many bacterial imprints on the surface of carbonate
of calcium along with small amounts of silica, magnesium, crystals, which suggested that bacteria might serve as
aluminum, potassium, iron, and titanium. XRD analysis nucleation sites for carbonate precipitation, which is in
showed that crystals formed by all bacteria were either agreement with earlier research works [29]. The bacterial
calcite and/or vaterite. Crystals precipitated by B. megaterium cell surface with various ions could nonspecifically induce
showed that the calcite phase formed the major form of mineral deposition by providing nucleation sites. Lian et al.
crystals along with a few vaterites. In the case of B. cereus [16] demonstrated that the process of carbonate crystal
and B. subtilis, the major phase was calcite along with very formation by Bacillus megaterium involved the nucleation
few vaterite peaks. B. thuringiensis showed vaterite as the of calcite on the bacterial cell walls. In the bacterial culture
major form of crystals along with a few calcite peaks, medium, Ca2+ is not likely utilized by bacterial metabolic
 BIOMINERALIZATION OF BACTERIAL CALCITE 713

processes; it just accumulates outside the cell. As a result inorganic components to specific crystallographic planes
of enzymatic reversible hydration of CO2, HCO3– or CO2 is of the growing crystal [15, 24].
produced and the dissolved CO2 transforms to CO23– or As CaCO3 crystals have advantages in special structures
HCO3– around the cell, commencing the growth of CaCO3 and excellent material properties for their use in various
crystals around the cell. industries like paper, paint, plastics, and medicines, along
XRD and FTIR studies showed the presence of calcite with bioremediation of building materials, calcium carbonate
as the main polymorph in three isolates, whereas two isolates polymorph selection can have important technical implications
had vaterite as the main phase. Vaterite is metastable at [18]. In conclusion, the present study has clearly shown
normal temperature and atmospheric pressure, and it has that strain-specific precipitation of various calcium carbonates
been suggested that metastable polymorphs form initially from bacterial isolates of calcareous soils occur during
and subsequently convert to stable the polymorph, calcite microbial CaCO3 precipitation, and, based on the type of
[12]. Despite extensive studies on bacterial carbonatogenesis, polymorph precipitated, this technology can be applied for
little is known on what the cause(s) of polymorph selection various purposes.
is during bacterial calcium carbonate mineralization. One
line of thought suggests that the phase and morphology of
calcium carbonate are bacterial (or strain)-specific [12]. It Acknowledgments
has also been suggested that specific proteins in EPS
produced by different bacteria types control aragonite or The authors are thankful to TIFAC-CORE, Thapar University,
calcite polymorph selection [15]. Braissant et al. [3] have Patiala, Punjab, India for provided lab facilities for research.
associated polymorph selection (vaterite vs. calcite) during We would also like to thank the Council of Scientific and
bacterial carbonatogenesis with the characteristics of EPS. Industrial Research (CSIR) for their financial support to
EPS proteins may specifically bind calcium ions and this project (37(1484)/2011/EMR-II). We also acknowledge
promote carbonate precipitation allowing right environmental Mr. Ashok Kumar Sahu from the Advanced Instrumentation
conditions, such as alkaline pH and the specific ion co- Research Facility (AIRF), JNU, for providing instrumental
ordination, being a nucleation site [9]. However, Ercole et support for the electron microscopy and confocal laser
al. [10] showed that EPS isolated from B. firmus and B. scanning microscopy.
sphaericus induce the precipitation of calcite. Tourney and
Ngwenya [31] indicated that dissolved organic carbon
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