Biomineralization of Calcium Carbonate Polymorphs ...

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Jan 27, 2013 - Microbially induced calcium carbonate precipitation. (MICCP) is a naturally occurring biological process that has various applications in ...
J. Microbiol. Biotechnol. (2013), 23(5), 707–714 http://dx.doi.org/10.4014/jmb.1212.11087 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 (MICCP) is a naturally occurring biological process that has various applications in remediation and restoration of a range of building materials. In the present investigation, five ureolytic bacterial isolates capable of inducing calcium carbonate precipitation were isolated from calcareous soils on the basis of production of urease, carbonic anhydrase, extrapolymeric substances, and biofilm. Bacterial isolates were identified as Bacillus megaterium, B. cereus, B. thuringiensis, B. subtilis, and Lysinibacillus fusiformis based on 16S rRNA analysis. The calcium carbonate polymorphs produced by various bacterial isolates were analyzed by scanning electron microscopy, confocal laser scanning microscopy, X ray diffraction, and Fourier transmission infra red spectroscopy. A strainspecific precipitation of calcium carbonate forms was observed from different bacterial isolates. Based on the type of polymorph precipitated, the technology of MICCP can be applied for remediation of various building materials. Key words: Calcite, vaterite, urease, Bacillus, biofilm, carbonic anhydrase

Biomineralization is the process of synthesis of inorganic mineral-like materials by living organisms. Calcium carbonate precipitation is a well-known example of extracellular bacterial biomineralization, and a large number of bacterial genera have been observed in a range of natural environments. Bacterial mineralization of calcium carbonates has found applications in the remediation (fixation) of metalcontaminated soil and groundwater [33], atmospheric CO2 sequestration [20], strengthening and consolidation of sand, limestone monument repairs, reduction of permeability of cement mortar, pores and cracks filling in concrete, and enhancing the strength of ash bricks [7, 8]. *Corresponding author Phone: +911752393743; Fax: +911752393738; E-mail: [email protected]

The precise role of microbes in the carbonate precipitation process is still not clear. However, three hypotheses that seem to be responsible for the process of carbonate crystallization are as follows: (i) mineralization occurs as a by-product of microbial metabolism involving either autotrophic or heterotrophic pathways [4]. During these passive processes, reactions, such as enzymatic hydrolysis of urea or the dissimilatory reduction of nitrate and sulfate, cause an increase in pH that shifts the bicarbonatecarbonate equilibrium toward the production of more CO32_ and ultimately leads to the precipitation of CaCO3, if free Ca2+ is present, (ii) nucleation of the carbonates take place on the cell wall, either due to ion exchange through the cell membrane [5] following some still poorly known mechanisms, or due to the support of negatively charged specific cell wall functional groups that adsorb divalent cations, such as Ca2+ [24], (iii) microbial extracellular polymeric substances (EPS) seem to play an important role in precipitation, either through the trapping and concentration of calcium ions or as a result of specific proteins that 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 presence of several negatively charged groups at neutral pH favors to bind to positively charged metal ions on bacterial surfaces, favoring heterogenous nucleation [2]. Biomineralization of calcium carbonate results in the production of different phases of calcium carbonate as anhydrous polymorphs: calcite, aragonite, and vaterite or two hydrated crystalline phases, monohydrocalcite (CaCO3·H2O) and ikaite (CaCO3·6H2O), but calcite and vaterite are the most common bacterial calcium carbonate polymorphs [25]. Besides its scientific interest, different calcium carbonate polymorphs have important technical implications, as in the case of the bacterial conservation of building materials, where the formation of coherent, durable calcium carbonate is required [18]. The same principle applies for other purposes like soil strengthening, cement and concrete protection,

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solid phase capture of groundwater metal contaminant, and/or the effective (long-term) CO2 sequestration via bacterial calcium carbonate mineralization. There is currently great interest in the development of materials based on biomineralization, but still very few bacteria have been exploited. In the present investigation, we isolated and characterized calcifying bacterial strains from calcareous soils and tested their abilities to produce urease, carbonic anhydrase, extra polymeric substances, biofilm, and calcium carbonate precipitates. These strains were identified based on biochemical and molecular characteristics. The properties of the crystals produced were analyzed morphologically and also for elemental composition. We aim to characterize the crystal aggregates precipitated by these bacterial isolates. The implications of these findings in understanding microbially induced calcium carbonate precipitation (MICCP) as well as its applications in building materials are discussed.

MATERIALS AND METHODS Isolation and Characterization of Bacteria Calcareous soil samples (pH 11.0) were collected from Anantapur District, Andhra Pradesh, India and the bacteria were isolated by the serial dilution method by plating the samples on urea agar base (Hi Media, India), a urease selective medium. Five isolates, designated as SS3, SS5, SS13, SS15, and SS18, were selected for further studies based on their abilities to produce urease qualitatively. The biochemical characterization of these isolates was done as per the standard protocols [13] along with morphological and physiological studies. Genomic DNA was extracted from overnight grown cultures, and 16S rRNA genes were amplified as described in Karn et al. [14]. The amplicons were purified with the QIA gel extraction kit (Qiagen, USA), and ligated into the pTZ57R/T vector as per the manufacturer’s instructions (Fermentas, USA). Ligated plasmids were transformed into Escherichia coli DH5α cells. Recombinant plasmids were sequenced using an Applied Biosystems automated DNA sequencer (DNA Sequencing Facility, Delhi University, India). BLAST analysis was performed to compare the sequences with available DNA sequences of the NCBI. The sequences were aligned using the MAFFT (http://mafft.cbrc.jp/alignment/server/) program and the alignment was manually corrected and a phylogenetic tree was constructed by the neighbor-joining method using MEGA 5.0 software [30]. The 16S rRNA gene sequences determined in this study are deposited in the GenBank of the NCBI under the accession numbers KC121060 to KC121064. These cultures were deposited at Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India. 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 chloride (NiCl2) along with 2% urea, and for carbonic anhydrase (CA) assay, 10 µM zinc sulfate (ZnSO4) was supplemented. The 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 assay was carried out as per the phenol-hypochlorite assay method

as described in Achal et al. [1]. One unit of urease is defined as the amount of enzyme hydrolyzing 1 micromole urea/min/ml. The carbonic anhydrase assay was performed as described by Smith and Ferry [26]. One unit of carbonic anhydrase activity is defined as the amount of enzyme required to form one µmole of p-nitrophenol per minute. Extracellular polymeric substances (EPS) production was determined according to the procedure described by Friedman et al. [11]. Biofilm formation was established aseptically in nutrient broth containing urea-CaCl2 on glass plates (25 × 75 mm) using the crystal violet (CV) method described by Morikawa et al. [21]. Precipitation of Carbonate Crystals and Analysis All bacterial cultures were incubated at 37oC for 3 weeks in NB media along with 2% urea and 25 mM CaCl2 (pH 8.0). The precipitated carbonates were collected on Whatman No. 1 filter paper by filtration, washed with sterile distilled water, and air dried at 37oC for 48 h, weighed, and analyzed for its morphological and chemical constituents. 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% 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 series of graded ethyl alcohol. The SEM observation was done under the following analytical conditions: EHT = 20.00 kV, WD = 10-11 mm. Elemental analysis was done with an energy dispersive X-ray analyzer (Bruker AXS, Quan Tax 200). In order to determine the morphological features of different crystals by confocal scanning laser microscopy (CSLM), the fluoresecent dye CTC (5-cyano-2,3ditolyl tetrazolium chloride; Polysciences Inc.) was used. Before the observation, the crystals were fixed for 30 min with 4% paraformaldehyde-phosphate-buffered saline. The slides were flooded with freshly prepared 1.5 mM (final concentration) CTC solution. The stain solution was left to react overnight at room temperature in the dark, and the slides were stored at 20oC for 30 min to stop the reaction. Before visualization with a confocal scanning laser microscope (LSM 510 Meta; Carl Zeiss), the slides were covered with a cover slip and fixed with DPX mountant (Hi Media). The stain was excited at 453 nm by the use of a 590-nm long-pass filter. XRD spectras were obtained using an X’ Pert PRO diffractometer with a Cu anode (40 kV and 30 mA) and scanning from 3o to 60o. The components of the sample were identified by comparing them with standards established by the International Centre for Diffraction Data. Fourier transform infra red (FTIR) spectra were recorded on a Bruker Vertex 70 apparatus by the diffuse reflectance accessory technique. The spectra of the crystals were scanned in the range of 400-4,000 cm-1. All experiments were performed in triplicates. The data were analyzed by analysis of variance (ANOVA) and the means were compared with a Tukey’s test. All the analyses were performed using GraphPad Prism (5.0) software.

RESULTS Isolation and Identification of Bacteria Among all the isolated bacteria, five isolates (viz., SS3, SS5, SS13, SS15, and SS18) were selected based on their

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high urease, carbonic anhydrase, and carbonate precipitation. Biochemical characteristics showed that these bacterial isolates are Gram-positive. They are rod-shaped, and catalase, urease, and oxidase positive. All isolates are motile except SS15, which was nonmotile. Starch, casein, and gelatin hydrolysis were recorded with SS3, SS13 and SS15, whereas SS5 and SS18 hydrolyzed casein and esculin. Nitrate reduction was observed by SS3, SS15, and SS18. Acid production was observed with sucrose, glucose, arabinose, ONPG, and malonate by SS3, but only with sucrose, glucose, and mannitol by SS5, SS13, SS15, and SS18. Citrate was utilized by SS3 and SS5. SS3 and SS5 isolates showed growth in a pH range of 6.5-11.5, whereas the other isolates had a pH range of 6-10.5. SS3 and SS18 were able to survive 0-8% NaCl, and the other isolates in the range of 0-7% NaCl. In the case of temperature tolerance, both SS3 and SS5 were able to survive in the range of 25-50oC, SS13 at 28-45oC, SS15 at 25-45oC, and SS18 at 25-55oC. The morphological, physiological, and biochemical characterization results showed marked

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differences among all the isolates. These isolates were further identified by 16S rDNA sequence analysis. BLAST analysis revealed that SS3 had 99% similarity (100% coverage) with Bacillus megaterium, SS5 had 99% similarity (100% coverage) with B. cereus, SS15 had 99% similarity (99% coverage) with B. thuringiensis, SS13 had 96% similarity (100% coverage) with B. subtilis, and SS18 had 99% similarity (97% coverage) with Lysinibacillus fusiformis. Phylogenetic analysis also grouped all these isolates to phylum Firmicutes and family Bacillaceae (Fig. 1). Urease and Carbonic Anhydrase Activities Among the isolates, Bacillus megaterium showed maximum urease activity, followed by B. thuringiensis, B. cereus, L. fusiformis, and B. subtilis (Fig. 2). The maximum urease activity observed by B. megaterium was 690 U/ml, followed by B. thuringiensis (620 U/ml) on the 4th day, while B. cereus, L. fusiformis, and B. subtilis produced 587, 525, and 515 U/ml on the 5th day, respectively. Carbonic anhydrase (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.

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Fig. 2. Urease and carbonic anhydrase activities of B. megaterium (Bm), B. cereus (Bc), B. thuringiensis (Bt), B. subtilis (Bs), and L. fusiformis (Lf) (values are mean ± SD).

compared with the other isolates (Fig. 2). The maximum CA activity was 115 U/ml by B. megaterium, followed by B. cereus (90 U/ml) on the 4th day, followed by 85, 70, and 60 U/ml by B. thuringiensis, B. subtilis, and L. fusiformis on the 5th day, respectively. EPS and Biofilm Production B. megaterium was able to produce 36.4 nmol/ml of EPS, followed by B. thuringiensis at 33.5 nmol/ml, L. fusiformis at 28.6 nmol/ml, B. subtilis at 26.8 nmol/ml, and B. cereus at 26.4 nmol/ml. B. megaterium also produced higher monoxenic biofilm (283 CFU/mm2) compared with B. cereus (243 CFU/mm2), B. thuringiensis (232 CFU/mm2), L. fusiformis (198 CFU/mm2), and B. subtilis (194 CFU/mm2) (Fig. 3). Carbonate Crystals Analyses Carbonate crystals were precipitated by all the isolates (Fig. 4). Carbonate crystals precipitated by B. megaterium

Fig. 3. Extracellular polymeric substances (EPS) and biofilm production by B. megaterium (Bm), B. cereus (Bc), B. thuringiensis (Bt), B. subtilis (Bs), and L. fusiformis (Lf) (values are mean ± SD).

Fig. 4. Calcium carbonate crystals precipitated by different bacterial isolates after 3 weeks (values are mean ± SD).

were highest over a period of three weeks at 37oC. B. megaterium produced 187 mg/100 ml, followed by B. subtilis (178 mg/100 ml), B. thuringiensis (167 mg/100 ml), B. cereus (156 mg/100 ml), and L. fusiformis (152 mg/100 ml). No crystals precipitated in the control sets. SEM studies of various crystals formed by different bacterial isolates showed obvious differences in size and shape (Fig. 5). The size of crystals formed by B. megaterium were 30-50 µm in diameter, whereas the size varied from 15-40 µm in B. cereus, 10-50 µm in B. subtilis, 2-15 µm in B. thuringiensis, and 2-10 µm in L. fusiformis. Crystals formed by B. megaterium were spherical, oval, rhombohedral as well as triangular in shape with smooth and rough surfaces. The number of crystals with smooth surfaces was relatively more abundant than with rough surfaces. Rodshaped imprints of bacteria were clearly observed on the surface of crystals (Fig. 5b). SEM observation also showed the formation of a mucous matrix over the calcified 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. 6). Polycrystal formation was observed in the case of B. megaterium. The crystals produced by other isolates appeared as separate and a mixture of spherical, ellipsoidal,

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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).

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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

Fig. 6. Confocal laser scanning electron microscopic images of bacterial carbonate crystals produced by (a) B. megaterium (Bm), (b) B. cereus (Bc), (c) B. thuringiensis (Bt), (d) B. subtilis (Bs), and (e) L. fusiformis (Lf).

and rectangular shapes. Bacterial cells seemed to be linked to the crystals. EDX analysis of all the isolates showed a large amount of calcium along with small amounts of silica, magnesium, aluminum, potassium, iron, and titanium. XRD analysis showed that crystals formed by all bacteria were either calcite and/or vaterite. Crystals precipitated by B. megaterium showed that the calcite phase formed the major form of crystals along with a few vaterites. In the case of B. cereus and B. subtilis, the major phase was calcite along with very few vaterite peaks. B. thuringiensis showed vaterite as the major form of crystals along with a few calcite peaks,

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 has been described to influence calcium carbonate precipitation in a positive way [15]. Along with EPS, the 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]. SEM and CLSM analyses indicated different morphologies of CaCO3 crystals induced by different bacteria. There were many bacterial imprints on the surface of carbonate crystals, which suggested that bacteria might serve as nucleation sites for carbonate precipitation, which is in agreement with earlier research works [29]. The bacterial cell surface with various ions could nonspecifically induce mineral deposition by providing nucleation sites. Lian et al. [16] demonstrated that the process of carbonate crystal formation by Bacillus megaterium involved the nucleation of calcite on the bacterial cell walls. In the bacterial culture medium, Ca2+ is not likely utilized by bacterial metabolic

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processes; it just accumulates outside the cell. As a result of enzymatic reversible hydration of CO2, HCO3– or CO2 is produced and the dissolved CO2 transforms to CO23– or HCO3– around the cell, commencing the growth of CaCO3 crystals around the cell. XRD and FTIR studies showed the presence of calcite as the main polymorph in three isolates, whereas two isolates had vaterite as the main phase. Vaterite is metastable at normal temperature and atmospheric pressure, and it has been suggested that metastable polymorphs form initially and subsequently convert to stable the polymorph, calcite [12]. Despite extensive studies on bacterial carbonatogenesis, little is known on what the cause(s) of polymorph selection 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 has also been suggested that specific proteins in EPS produced by different bacteria types control aragonite or calcite polymorph selection [15]. Braissant et al. [3] have associated polymorph selection (vaterite vs. calcite) during bacterial carbonatogenesis with the characteristics of EPS. EPS proteins may specifically bind calcium ions and promote carbonate precipitation allowing right environmental conditions, such as alkaline pH and the specific ion coordination, being a nucleation site [9]. However, Ercole et al. [10] showed that EPS isolated from B. firmus and B. sphaericus induce the precipitation of calcite. Tourney and Ngwenya [31] indicated that dissolved organic carbon (DOC) released from EPS produced by B. liqueniformis complexes Ca ions, reducing the calcium carbonate saturation and favoring calcite precipitation over vaterite. EPS also contribute to the stability of biofilms and have been reported to influence the polymorphic development of CaCO3 during mineralization [32] by capturing the produced calcium carbonate, which might result in a homogeneous layer of calcium carbonate. These observations may imply that carbonatogenic bacteria can exert a higher degree of control on biomineralization than previously thought, as morphology and polymorph selection are characteristics of bacterially controlled mineralization in higher order organisms [17]. It has also been reported that the culture medium includes several organic molecules whose incorporation is a general phenomenon during calcium carbonate biomineralization. It has been seen that the incorporation of such organic molecules has profound effects on the morphology of precipitates [6]. Morphological variations in crystals produced by different bacteria were observed in this study, suggesting variations in crystal growth rates along different planes of the crystal structure. This could have been a result of the colony growth rate and/or actual urease activity, which thus influenced the rate of supply of chemical species required for precipitation [27]. Alternatively, crystal growth can be inhibited or altered by the adsorption of proteins, organic matter, or

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inorganic components to specific crystallographic planes of the growing crystal [15, 24]. As CaCO3 crystals have advantages in special structures and excellent material properties for their use in various industries like paper, paint, plastics, and medicines, along with bioremediation of building materials, calcium carbonate polymorph selection can have important technical implications [18]. In conclusion, the present study has clearly shown that strain-specific precipitation of various calcium carbonates from bacterial isolates of calcareous soils occur during microbial CaCO3 precipitation, and, based on the type of polymorph precipitated, this technology can be applied for various purposes.

Acknowledgments The authors are thankful to TIFAC-CORE, Thapar University, Patiala, Punjab, India for provided lab facilities for research. We would also like to thank the Council of Scientific and Industrial Research (CSIR) for their financial support to this project (37(1484)/2011/EMR-II). We also acknowledge Mr. Ashok Kumar Sahu from the Advanced Instrumentation Research Facility (AIRF), JNU, for providing instrumental support for the electron microscopy and confocal laser scanning microscopy.

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