Appl Biochem Biotechnol (2014) 173:307–317

DOI 10.1007/s12010-014-0842-1

Influence of Calcium Sources on Microbially Induced

Calcium Carbonate Precipitation by Bacillus sp. CR2

Varenyam Achal & Xiangliang Pan

Received: 14 January 2014 / Accepted: 27 February 2014 /

Published online: 19 March 2014
# Springer Science+Business Media New York 2014

Abstract Stimulation of microbially induced calcium carbonate precipitation (MICCP) is

likely to be influenced by calcium sources. In order to study such influences, we performed
MICCP using Bacillus sp. CR2 in nutrient broth containing urea, supplemented with different
calcium sources (calcium chloride, calcium oxide, calcium acetate and calcium nitrate). The
experiment lasted 7 days, during which bacterial growth, urease activity, calcite production and
pH were measured. Our results showed that calcium chloride is the better calcium source for
MICCP process, since it provides higher urease activity and more calcite production. The
influences of calcium sources on MICCP were further studied using Fourier transform-infrared
(FTIR) spectroscopy, scanning electron microscopy (SEM) and X-ray diffraction (XRD)
analyses. These analyses confirmed that the precipitate formed was CaCO3 and composed
of predominantly calcite crystals with a little amount of aragonite and vaterite crystals. The
maximum yield of calcite precipitation was achievable with calcium chloride followed by
calcium nitrate as a calcium source. The results of present study may be applicable to media
preparation during efficient MICCP process.

Keywords Bacillus . Calcite . Aragonite . Urease . Calcium chloride

Introduction

Microbially induced calcium carbonate precipitation (MICCP) plays an important role in the
remediation of toxic metals, including radionuclides, wastewater treatment, soil improvement
and carbon sequestration [1–6]. The importance of MICCP has also been widely recognized in
geotechnological and structural engineering, especially remediation of cracked concrete,
conservation of monuments, sealing porosity or rock fractures and enhancement in the
durability of concrete structures [1, 7–10].
A wide range of microorganisms carry out calcite precipitation in the environment by
producing an enzyme, urease, hydrolyzing urea to produce CO32− ions [11]. Calcite precipi-
tation in solution occurs via the overall equilibrium reaction of Ca2 + +CO23 − ⇔CaCO3.
Because the production of CO32− from bicarbonate (HCO3−) in water is strongly pH-

V. Achal : X. Pan (*)

Laboratory of Environmental Pollution and Bioremediation, Xinjiang Institute of Ecology and Geography,
Chinese Academy of Sciences, Urumqi 830011, China
e-mail: xlpan@ms.xjb.ac.cn
308 Appl Biochem Biotechnol (2014) 173:307–317

dependent, an increase in CO32− concentration occurs under alkaline conditions. Therefore,

calcium carbonate precipitation readily occurs in alkaline environments abundant in calcium
(Ca2+) and carbonate (CO32−) ions [12–14]. It was hypothesized that every bacterial cell could
serve as a nucleation site for CaCO3 precipitation when the cell density was low [14]. Possible
biochemical reactions in the nutrient media inoculated with the bacteria to precipitate calcium
carbonate at the cell surface are presumed as follows:

Ca2þ þ Cell→Cell−Ca2þ
HCO−3 ↔H þ þ CO2−
3
Cell−Ca2þ þ CO2−
3 ↔Cell−CaCO3

Microbial calcite precipitation is known to be conducted by a wide range of bacterial species in

an alkaline environment rich in Ca2+ ions [12–16]. The precipitation of calcite by bacteria such as
Bacillus megaterium has been described as involving stoichiometric interactions between positively
charged Ca2+ ions and the negatively charged bacterial cell wall [17, 18]. Such interaction produces
changes in the overall charge of the cell wall, leading to an increase in the size of biomineral formed
by bacteria. Extracellular polymeric substances (EPS) produced by bacteria have also been shown to
be involved in the process of biocalcification by entrapping Ca2+ and serving as a nucleation site
[19]. The availability of a nucleation site is very important for continuous and stable calcium
carbonate formation. It is isolated from the environment by a delimiting geometry by limiting the
diffusion in and out of the system [9]. EPS adsorb Ca2+ continually so that carbonate microcrystals
form on the surface of the biofilm and result in calcification.
One of the most acceptable hypotheses for calcium carbonate precipitation is that calcium
ions are not used in microbial metabolism, and hence accumulate in the extracellular medium
[17]. Calcium sources might have a tremendous role in MICCP process; however, previous
studies mainly focused on the physiological basis for MICCP and saturation state of the water
with respect to calcium carbonate minerals [17, 20]. Furthermore, MICCP process is widely
used in bio-civil engineering to improve the durability of building materials. As bacteria
function as the catalyst, a suitable mineral precursor compounds as calcium needs additionally
to be incorporated in the material matrix to provide a truly autonomous healing mechanism in
building structures. The deposition of calcium carbonate to improve the durability of building
materials is influenced by the ureolytic bacteria and calcium salt [21]. In order to study the
influence of calcium sources, calcite precipitation studies have been carried out using a
bacterium, Bacillus sp. CR2, in the present investigation.
The influence of calcium sources on the MICCP process by Bacillus sp. CR2 was assessed
on the basis of bacterial growth, urease activity, calcite production and pH profile. Fourier
transform-infrared (FTIR) spectroscopy was employed to analyze the vibrational modes in
carbonate mixtures induced by bacterial cells with different calcium sources. Scanning electron
microscopy (SEM) and X-ray diffraction (XRD) analyses evidenced the influence of calcium
sources on MICCP.

Materials and Methods

Bacterium and Culture Media

The urease producing microorganism used in present study was Bacillus sp. CR2, isolated
from mine tailing soil of Urumqi, Xinjiang, China. This isolate was identified based on detail
molecular analyses using 16S rRNA gene sequencing. 16S rRNA gene sequence was
Appl Biochem Biotechnol (2014) 173:307–317 309

deposited in GenBank of NCBI under the accession number HQ331531. The bacterium was
cultured in Nutrient broth, which contains (per liter of deionized water): peptone 10 g, yeast
extract 1.5 g, beef extract 1.5 g, NaCl 5 g.
To study the microbial calcite precipitation induced by Bacillus sp. CR2, the Nutrient broth
was supplemented with 2 % urea to accelerate urease production. Further to induce calcite
precipitation, different calcium sources viz. calcium chloride, calcium oxide, calcium acetate
and calcium nitrate at the concentrations of 25, 50, 25 and 40 mM, respectively, were
amended, separately, in the media. The particular concentration of calcium source was
optimized to produce maximal urease in nutrient broth containing 2 % urea by Bacillus sp.
CR2. The final pH of the medium was adjusted to 7.5 and growth condition was provided at
30ºC. The control experiments were simulated in similar way, but without addition of any
calcium source in the nutrient media.

Bacterial Growth and Urease Production

Bacterial growth was determined by recording the cfu (colony forming unit) count in the Nutrient
broth supplemented with urea and calcium source at an interval of 24 h up to 7 days under shaking
condition (130 rpm). The pH was also measured during this experiment at regular time interval.
The urease production by Bacillus sp. CR2 was measured in said broth media by measuring
the amount of ammonia released from urea according to the phenol–hypochlorite assay
method at different time intervals as described by Achal et al. [22]. One unit of urease is
defined as the amount of enzyme hydrolyzing 1 μmol urea/min.

Calcite Production

The calcite precipitated by Bacillus sp. CR2 in the Nutrient broth supplemented with urea and
calcium source was determined at regular time intervals for 7 days. Separation of the bacterial
cell and calcite in the culture broth was made by filtering through 0.45-μm Whatman membrane
filter papers; the calcite was retained on the membrane and the bacterial cell in the filtrate [23].
Calcite production was expressed as calcite dry weight (mg)/cell dry mass (mg).

FTIR Spectroscopy

To evaluate the calcium carbonate precipitation by Bacillus sp. CR2, infrared spectroscopic
analysis was performed with an FTIR spectrometer (Tensor 27 FTIR; Bruker, Germany). After
growing the bacterial cells in different calcium sources for 7 days, the samples were lyophilized to
make it into powder form. About 1 mg of cells (dry weight) was mixed and ground with 100 mg
of KBr in an agate mortar. Pure calcium carbonate was used as control to identify CaCO3
minerals. The FTIR spectra obtained at 4,000–400 cm−1 were used to examine the cell pellet.

XRD Analysis

The calcite induced by Bacillus sp. CR2 in media containing different calcium sources was
analyzed by XRD to confirm biomineralization. XRD spectra were obtained using Bruker D8
diffractometer with a Cu anode (40 kV and 30 mA) and scanning from 5° to 80° 2θ. All the
samples were lyophilized prior to XRD analyses and the samples were grinded using motor
pestle before mounting on to a glass fiber filter using a tubular aerosol suspension chamber.
The components of the samples were identified by comparing them with standards established
by the International Center for Diffraction data.
310 Appl Biochem Biotechnol (2014) 173:307–317

SEM Analysis

Calcium carbonate precipitation in different media was visualized by SEM. The grown
bacterial cells were harvested at the end of 7 days and sediments were dried at 45ºC. Samples
were gold coated with a sputter coating Emitech K575 prior to examination under SEM (Zeiss
SUPRA 55VP, Germany) and examined at accelerating voltages ranging from 10 to 25 kV.

Statistical Analysis

All the experiments were performed in triplicates. Error bars on graphs show the standard
deviation. The data were analyzed by analysis of variance (ANOVA) and the means were
compared by Tukey’s test (p<0.05) using the GraphPad Prism software (version 5.0).

Results and Discussion

Effect of Calcium Sources on Bacterial Growth and pH

The growth profile of Bacillus sp. CR2 was studied up to 168 h in urea containing nutrient
media supplemented with different calcium sources. The growth of bacterial species was
noticed to increase in all the media till 48 h and remained in stationary phase up to 120 h
(Fig. 1a). There was no significant difference in cfu among different media; however, the
maximum growth was observed in media containing calcium chloride while calcium oxide
was the least preferred calcium source to grow the bacterial species (Fig. 1a). The log cfu/ml
was marginally higher with calcium nitrate compared to the calcium acetate.
The pH of the media containing different calcium sources significantly increased with the
increase in growth of bacterial isolate; however, the pH of the control medium was almost
similar till the end of the experiment. The pH data obtained confirmed the process of urea
hydrolysis in calcium containing media (Fig. 1b). Initially, at 24 h the pH was 8.1 in the
medium containing calcium oxide, while it was 8.6 with calcium acetate. The pH increased up
to 9.3 and 9.1 in the media containing calcium chloride and calcium nitrate, respectively, by
Bacillus sp. CR2. With a continuous increase in pH, carbonates precipitate with calcium to
form calcite indicated by urease production (Fig. 2b). This can be explained that ureolysis
leads to an increase in pH and a shift in the bicarbonate equilibrium. Urea hydrolyzing bacteria
can promote calcium carbonate precipitation by hydrolyzing urea and producing ammonium
and bicarbonate ions, thereby increasing the pH [24]. The generation of NH4+ increases local
pH and the reaction continues spontaneously to form calcium carbonate in the presence of
calcium ions and the availability of nucleation sites [5, 13]. The maximum pH was observed at
120 h and decreased thereafter irrespective of calcium sources used. With prolonged incubation,
ammonia production may be less in all the media, leading to drop in pH value after 120 h. pH
drops when the rate of calcium carbonate precipitation is close to its maximum. The incorpo-
ration of Ca2+ and CO32− in the newly formed carbonates brings about the pH reduction [25].

Urease and Calcium Carbonate Productions

Bacillus sp. CR2 showed the maximum urease activity in the medium containing calcium
chloride, although urease production profile was similar irrespective of calcium sources used.
After 120 h, urease activity was decreased in all the media. It has been reported that a high
level of protease production after 120 h may have inhibitory effect on urease production and
Appl Biochem Biotechnol (2014) 173:307–317 311

(a)
9.0
Control
Ca chloride Ca nitrate Ca oxide Ca acetate
7.5

6.0
log cfu mL-1

4.5

3.0

1.5

0.0

(b)
11

10
pH

7
0 24 48 72 96 120 144 168
Time (h)
Fig. 1 The a evolution of the cfu in time, and b pH profile of Bacillus sp. CR2 cells in nutrient media containing
different calcium sources and compared with control

leads to the possibility of urease degradation [15]. The maximum urease produced with
calcium chloride was 432 U ml−1 followed by 418 U ml−1 with calcium nitrate (Fig. 2a).
Bacillus sp. CR2 produced the lowest (p<0.05) amount of urease (389 U ml−1) in the medium
containing calcium oxide, while activity was recorded at 401 U ml−1 in the medium containing
calcium acetate. The results were in agreement with previous studies [11, 15, 19].
The calcite precipitation induced by Bacillus sp. CR2 was estimated in all media containing
different calcium sources. It was in correlation with urease production and the maximum
amount was recorded in the medium containing calcium chloride at the end of 120 h.
Negligible amount of calcite was precipitated in the medium where no calcium source was
added. Calcite estimation was expressed as calcite (mg)/dry cell mass (mg). Calcium chloride
was the most preferred calcium sources that produced significantly higher (P<0.05) amount of
calcium carbonate (2.32 mg) by Bacillus sp. CR2 (Fig. 2b). The minimum calcite amount
(1.84 mg) was precipitated in the medium containing calcium oxide, while 1.86 mg of calcite
312 Appl Biochem Biotechnol (2014) 173:307–317

(a)
500 Control
Ca chloride Ca nitrate Ca oxide Ca acetate
Urease activity (U mL-1)
400

300

200

100

(b)
2.5
Calcite (mg)/cell mass (mg)

2.0

1.5

1.0

0.5

0.0
0 24 48 72 96 120 144 168
Time (h)
Fig. 2 The a urease and b calcite productions by Bacillus sp. CR2 cells in nutrient media containing different
calcium sources and compared with control

was determined with calcium acetate. Lee [23] used Luria–Bertani medium containing 1.5 %
calcium acetate to estimate calcite by B. amyloliquefaciens CMB01 and found that the
maximized yield was 0.91 mg of calcite after 120 h [23].
Urease is one of the important enzymes that leads to calcium carbonate precipitation and has been
reported to produce significantly in the media containing urea and calcium [14, 19, 22]. Urea
hydrolyzing bacteria couple calcification to their metabolic assimilation processes to scavenge protons
[26]. The presence of ammonium ions and the additional release of CO2 into the surrounding medium
increase the pH that accelerates the rate of the urease induced calcium carbonate precipitation.

Calcium Carbonate Characterization by FTIR Spectroscopy

FTIR spectroscopy is used to analyze the composition of calcification during biomineralization

process [16]. The FTIR spectra of calcium carbonate precipitate in media with different
Appl Biochem Biotechnol (2014) 173:307–317 313

calcium sources are presented in Fig. 3. In the presence of calcium chloride, calcium nitrate,
and calcium oxide, the FTIR spectra showed formation of aragonitic mineral phase by
appearance of the band at 856 cm−1, while spectra coexisted with calcite (sharp band at
873 cm−1) in the media with all the four calcium sources (Fig. 3). The precipitates obtained
from Bacillus sp. CR2 cells gave rise to highly characteristic sharp stretching bands (weak in
case of the media containing calcium acetate and calcium oxide) of calcites at 1,800 to
1,550 cm−1 [27, 28]. The bands located in the range 3,600 to 3,400 cm−1 verified the
interaction that takes place on both the hydroxyl and amine groups, indicating the formation
of more –OH groups, although the bands were weak in the media containing calcium acetate
and calcium oxide [16, 29].
FTIR appeared to be a convenient method for early identification of calcium
carbonate crystals from bacteria in a liquid medium [27]. The predominant mineral
formed by Bacillus sp. CR2 cell was calcite (as inferred from the relative intensity of
the bands at 873 cm−1 and 1,800 to 1,550 cm−1), which were sharper in the media
containing calcium chloride and calcium nitrate and further confirms the capacity of
the such calcium sources to promote the precipitation of calcite.

Calcium Carbonate Characterization by XRD Analysis

To support the FTIR spectra for calcium carbonate precipitation induced by Bacillus sp. CR2
in the media with different calcium sources, the precipitates were further characterized by
XRD analysis. The XRD spectra showed that Bacillus sp. CR2 produced mainly calcite in the
media with all four different calcium sources; however, maximum number of calcite peaks was
observed in the medium containing calcium chloride (Fig. 4). Aragonite and vaterite peaks
were also observed in all culture media except in the media containing calcium acetate as the
calcium source. These two minerals are polymorph of calcium carbonate and among them

5.0

4.5 CaCO3
4.0 Ca acetate
Ca oxide
3.5 Ca chloride
3.0 Ca nitrate
Absorbance

2.5

2.0

1.5

1.0

0.5

0.0

-0.5
4000 3500 3000 2500 2000 1500 1000 500
-1
Wavenumber (cm )
Fig. 3 FTIR analysis confirming calcium carbonate precipitation induced by Bacillus sp. CR2 cells in media
containing different calcium sources along with pure CaCO3
314 Appl Biochem Biotechnol (2014) 173:307–317

vaterite is less stable and can be transformed into calcite on simple contact with water at room
temperature [30]. A sodium chloride (NaCl), an important constituent of the media, peak was
common among all treatments.
Calcium carbonate precipitation along with extra peaks of sodium nitrate (NaNO3) was
observed from the calcium nitrate-supplemented culture. This observation is explained by the
following equations.
Na2 CO3 ðaqÞ þ CaðNO3 Þ2ðaqÞ →CaCO3ðsÞ þ 2NaNO3ðaqÞ 2Naþ ðaqÞ þ CO3 2− ðaqÞ þ Ca2þ ðaqÞ þ 2NO3− ðaqÞ →CaCO3ðsÞ

þ 2Naþ ðaqÞ þ 2NO3− ðaqÞ Net Ionic : Ca2þ ðaqÞ þ CO3 2− ðaqÞ ↔CaCO3ðsÞ

Calcium oxide (CaO) was also found to be an effective calcium source in Bacillus sp. CR2
induced calcite precipitation, implied by the XRD analysis. The carbonation route is an
industrial route for calcium carbonate precipitation, which uses calcium hydroxide, also
observed in XRD spectra (made by hydration of CaO with water) and carbon dioxide for
precipitation [31]. The probable reactions are as shown below:
Hydration : CaO þ H 2 O→CaðOH Þ2
Precipitation : CaðOH Þ2 þ CO2 →CaCO3 þ H 2 O
It is clearly depicted from the XRD analysis that calcium chloride was the most preferred calcium
sources for calcium carbonate precipitation induced by Bacillus sp. CR2, which is consistent with
urease and calcite production. XRD analysis confirmed the presence of multiple mineral forms,
including aragonite and vaterite, which is only transiently found in nature. Our results indicate that
solution matrix containing calcium sources will influence the resultant morphologies of the precip-
itates such as aragonite and vaterite [32]. Aragonite and vaterite are relatively unstable polymorphs
of calcium carbonate and can be rapidly transformed into calcite in aqueous solution. Bacteria may
not be able to influence the mineralogy of the precipitates, but calcifying organic substances derived
from the decay of bacteria might trigger matrix-mediated precipitation [32]. Furthermore, the degree

Fig. 4 XRD spectra confirming calcium carbonate precipitation induced by Bacillus sp. CR2 cells in media
containing different calcium sources (C calcite, A aragonite, V vaterite, NaN sodium nitrate, CaH calcium
hydroxide)
Appl Biochem Biotechnol (2014) 173:307–317 315

Fig. 5 Scanning electron micrographs showing calcium carbonate crystals along with rod shaped Bacillus sp.
CR2 cells in media containing a calcium chloride, b calcium nitrate, c calcium acetate, d calcium oxide and e
control

of carbonate supersaturation predetermines the mineralogy of the precipitates, with very high
supersaturation levels promoting aragonite or vaterite formations [13, 33]. The Nutrient broth
medium containing calcium acetate might not achieve higher supersaturation level, thus no aragonite
or vaterite formed.

Calcium Carbonate Characterization by SEM Analysis

The FTIR and XRD analyses confirmed the presence of calcium carbonate crystals, which were
observed under SEM. SEM image illustrated the shapes of calcium carbonate crystals formed by
Bacillus sp. CR2 in the media containing calcium chloride, calcium nitrate, calcium acetate and
calcium oxide (Fig. 5). The presence of crystalline calcite associated with bacteria indicates that
bacteria served as nucleation sites during the mineralization process [14]. The clearest calcite
316 Appl Biochem Biotechnol (2014) 173:307–317

crystals along with bacterial cells were observed in the medium containing calcium chloride
(Fig. 5a), followed by calcium nitrate (Fig. 5b). The crystal like structures were also found to be
associated with calcium acetate and calcium oxide (Fig. 5c and d), which were absent in control
(Fig. 5e). Boquet et al. [34] showed that most soil bacteria are able to precipitate crystals of
calcium carbonate when tested in a medium containing calcium acetate, and that calcite produc-
tion by bacteria is just a function of the medium composition. Urzi et al. [35] found a variety of
crystal morphologies and big crystals of size up to 150 μm, produced by bacteria in medium
containing calcium acetate. The SEM results demonstrated the presence of mineral polymorphs,
including calcite, vaterite and aragonite [36]. Many of the calcite crystals also contained obvious
rod-shaped bacterial cells closely associated with, and emerging from, the mineral surface.

Conclusions

Previous studies showed that the composition of the culture medium, pH, and salinity can
change the type and amount of calcium carbonate. However, it is for the first time the detailed
investigation was carried out on the MICCP by different calcium sources. Estimation of
microbially induced calcium carbonate by the combination of FTIR spectroscopy, SEM and
XRD methods confirmed the influences of different calcium sources on it. Calcium chloride
was the most preferred calcium source for calcite precipitation followed by calcium nitrate. An
appropriate medium to grow bacterial cells during biocalcification process is of concern. These
results have important implications for the design of future engineering technologies involving
microbially induced calcite precipitation, such as sand consolidation, soil improvement,
remediation of cracked concrete and conservation of monuments.

Acknowledgements This work was supported by National Natural Science Foundation of China (U1120302,
41072195 and 41350110533). We are thankful to the editor and reviewers for their constructive comments for the
improvement of the manuscript.

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