Biosynthesis of Calcite Nanocrystal by a Novel Polyextremophile Bhargavaea cecembensis-Related Strain Isolated from Sandy Soil

Urease-producing bacteria are abundant in soils, which can precipitate calcium carbonate nanocrystals by enzymatic hydrolysis of urea in the presence of calcium ions. This process is known as microbially induced calcium carbonate precipitation (MICP), and it has received much attention in recent years as an eco-friendly technology. Therefore, the purpose of the present study was to isolate local extremophile bacterial strains capable of producing calcium carbonate. Among a total of 44 isolated urease-producing strains from sandy soils, one strain with a high level of urease activity (8.16 U/ml) and production of a large amount of calcium carbonate (410 mg/100 ml) was selected for further investigation. 16S rRNA gene sequencing showed that this strain had 99.66% sequence identity to Bhargavaea cecembensis. The SEM-EDX and XRD analyses indicated that irregular vaterite and aggregated nanocalcite were the dominant polymorphs produced by this strain. The size of these nanocalcite crystals ranged between 25 and 42 nm. The selected strain showed high levels of tolerance to different conditions of temperature, pH, and salinity. This strain grows at high temperatures up to 50 °C, alkaline pH (9–11), and high concentrations of NaCl (20–25% w/v). Flow cytometry analysis demonstrated 96% cell viability of the isolated strain after desiccation stress. Bhargavaea was first reported in 2009 as a new genus, and it belongs to the Firmicutes. So far, there has been no report on its MICP potential. The present study is the first one to report nanocrystal calcium carbonate precipitation in polyextremophile Bhargavaea cecembensis, which makes it a suitable candidate for bio-cementation under extreme circumstances.


Introduction
Biomineralization is the chemical change of an environment by the activity of living organisms [1]. This process is associated with the production of different kinds of bio-minerals [2]. These biological minerals are available in two categories: extra-cellular and intra-cellular inorganic crystals [1]. MICP is a well-known example of extra-cellular biomineralization [3]. MICP has been widely observed in aquatic and soil environments, and many microorganisms contribute to this process [4].
For the first time, Bouquet and his colleagues found that soil bacteria induce calcium carbonate mineralization through their metabolism [5]. This led to further research into the mechanisms involved in this biological process and its applications.
Bacteria can carry out MICP through several mechanisms, such as anaerobic sulfide oxidation, sulfate reduction, urea hydrolysis, and photosynthesis [6]. Among them, calcium carbonate production in soil with ureolytic bacteria is the most common method used in many related studies [7][8][9]. In this method, the bacterial urease enzyme hydrolyzes urea into ammonium and carbonate. This hydrolyzation results in the release of hydroxide and ammonium ions. Then, established ionic equilibrium contributes to an increase in the pH of the environment. Finally, the presence of calcium ions in the medium causes a reaction with produced carbonate anions, which is followed by calcium carbonate precipitation [10].
There are different crystalline phases of calcium carbonate, including three forms of anhydrous calcium carbonate (calcite, vaterite, and aragonite) and two hydrate crystalline polymorphs (monohydrocalcite, hexahydrocalcite, or ikaite). Among them, calcite and vaterite are stable and common forms [1,11]. The medium composition and bacterial type are the most important factors affecting these morphologies [12].
This bacterial-induced calcium carbonate mineralization is a cost-effective and eco-friendly technology. Therefore, it has been recently considered as a method for a broad range of applications, such as restoration of limestone, reducing permeability in cement, filling pores and cracks in concrete, and enhancing the strength of ash bricks [1,3,[13][14][15][16][17]; however, most of the above cases are relatively harsh environments for the growth and survival of microorganisms. For example, concrete has a rather dry matrix with alkaline pH, and it lacks essential nutrients for most bacteria, so that only extremophiles, such as bacterial species that are resistant to heat and desiccative environments, can withstand these harsh conditions and enhance the strength of concrete at the same time [18]. Due to this feature, the development of bio-cementation technology using such bacteria, which can precipitate calcium carbonate in extreme conditions, will be a valuable practice [19].
Therefore, the current study aimed to isolate and identify bacteria strains, which can produce calcium carbonate crystals, from the soil samples of Iranian desert regions and then to investigate their tolerance to extreme conditions.

Collection of Soil Samples
Fifteen sandy soil samples were aseptically collected from separate desert regions around three cities, including Mahshahr, Ahvaz, and Omidiyeh in Khuzestan Province, Iran (Fig. SI1). Samples were taken from a depth of 5 to 10 cm from the surface at all locations using a sterile spatula. They were placed in sterile polyethylene bags and transported to a laboratory at the University of Isfahan within 12 h.

Isolation and Screening of Urease-Producing Bacteria
To isolate urease-producing bacterial strains, 10 g of each soil sample was dissolved in 90 ml of sodium chloride solution 0.85%, and then, the suspensions were placed on an orbital shaker and mixed for 30 min. Then, 0.1 ml from each sample was spread in urea agar base (Christensen's medium) plates and incubated for 7 days at 30 °C. Bacterial growth and color changes in the medium were monitored each day during the incubation period [20]. Finally, urease-positive single colonies were streaked on new Christensen's media and screened for calcium carbonate crystal production after incubation at 30 °C for 7 days.

Investigation of Calcium Carbonate Crystal Production
Isolated urease-positive colonies were assayed for calcium carbonate production by streaking the culture on calcium carbonate precipitation (CCP) medium [21] (containing: NaHCO 3 2.12 g/l, NH 4 Cl 10 g/l, nutrient broth (N.B) 3 g/l, urea 20 g/l, CaCl 2 30 mM, and agar 20 g/l). Plates were incubated for 7 days at 30 °C and checked daily for crystal production. Some produced crystals were observed by using an optical microscope [22]. Finally, selected strains were inoculated into 250-ml glass Erlenmeyer flasks containing 100 mL of N.B medium, urea 2%, and CaCl 2 0.37 g. The culture media were incubated at 30 °C under aerobic conditions for 14 days and checked every day for calcium carbonate crystals precipitation. Eventually, the crystals accumulated at the bottom of the Erlenmeyer were collected on a Whatman filter paper and weighed after complete oven-drying at 70 °C [23].

Characterization of Produced Crystals by XRD, TEM, and SEM-EDX
The calcium carbonate crystals collected using Whatman paper in the previous step were ground into powder. Then, the XRD method (D8ADANCE, Beuker, Germany) was used to determine the crystallographic structure and polymorphism of the powders. Samples were scanned in the 2θ angle range of 5 to 80°, which the maximum possible angle was 90°.
Their morphologies were observed using SEM (TES-CAN, MIRA3) and TEM (CM120, Holland). Samples for SEM and TEM were prepared by their dispersion using the ultrasonication technique. Then, every sample was coated with gold before SEM and put on a Cu grid before TEM imaging.
Chemical components of the gathered powders were characterized using EDS elemental content maps [24].

Molecular Identification of the Selected Strains
Biochemical tests were used for phenotypic identification of the isolates based on standard procedures. Finally, some of them were selected for molecular identification, which includes the following steps:

Amplification of 16S rRNA Gene by PCR
Selected strains were identified using 16S rRNA gene amplification with 27F-YM as universal forward and 1492R as universal reverse primers [25,26]. Ready Master Mix (Cin-naGen, Iran) was applied for the preparation of PCR solution, which contained 10× PCR buffer, dNTP, MgCl 2, and Taq DNA polymerase. Then, samples were placed in a thermocycler (Eppendorf, Germany), which was set to an initial denaturation at 95 °C for 5 min, followed by 24 cycles of 45-s denaturation at 94 °C, 1 min annealing at 55 °C, 1 min extension at 72 °C, and a final extension at 72 °C for 7 min. PCR products were run on 1% agarose gel electrophoresis, and the bands were observed after ethidium bromide staining under a UV lamp. Finally, PCR products were sequenced by FazaBiotech Co. (Tehran, Iran).

Phylogenetic Analysis
The 16S rRNA sequences of isolated strains were blasted by the Nucleotide Blast program in NCBI databases to find homologous sequences. After importing 16S rRNA sequences of our strains and other close species, alignment and drawing the phylogenetic tree were performed using Mega.10 software with Clustal W and Neighbor-joining methods, respectively. Bootstrap values with 1000 replications were used to calculate the confidence levels of clades of the phylogenetic tree [27].

Quantitative Measurement of Urease Activity
Urease activity was assayed by the colorimetric method. First, the isolated strains were cultured overnight to prepare a cell suspension with an optical density of 0.5 at 600 nm by phosphate-buffered saline (PBS). Twenty microliters of each suspension was inoculated into 200 uL Stuart's urea broth (SB, containing: 0.1 g/l yeast extract, 9.5 g/l Na 2 HPO 4 , 9.1 g/l KH 2 PO 4 , 20 g/l urea, and 0.01 g/l phenol red) in triplicate on a 96-well microtiter plate. Finally, the plate was incubated for 24 h at 28 °C and the optical density of each well was checked at 550 nm every hour using a microplate reader (Bio-Rad, USA). Negative controls were non-inoculated SB media. Enzyme urease (Jack beans, Sigma) was also used to create a standard curve for urease activity (U/ml). In this method, one unit (U) of urease activity is equivalent to the amount of enzyme that hydrolyzes 1 μM of urea per minute [28,29].

Effect of Temperature, Salinity, and pH on the Growth of the Selected Strain
To study the effect of temperature on bacterial growth, the selected strain was grown in N.B. Then, the turbidity of bacterial suspension was adjusted to 0.5 McFarland. One percent of this suspension was inoculated in N.B medium, and the flasks were incubated at various temperatures (20-50 °C) for 10 days. Finally, the bacterial growth rate was determined by turbidity measurement of culture at 600 nm using a spectrophotometer (APEL PD-303). To assess the effect of pH on bacterial growth, a pH range of 3 to 11 was considered. These solutions were prepared with 1 N NaOH and 1 N HCl. The effect of salt was studied in the NaCl concentration range of 0 to 25% (w/v). Both tests were performed according to the method mentioned for the temperature factor [30].

Determination of the Selected Strain Resistance to Desiccation Stress
Bacterial tolerance to desiccation was determined using a 96-well microtiter plate assay. First, cell suspension equivalent to A600 nm = 0.5 was prepared from the late log-phase culture of the selected strain. Then, 0.2 ml of each suspension was added to each well of the 96-well microtiter plate. Finally, the plate was covered and kept for 4 weeks at 30 °C. After this period, the content of each well was completely dissolved in 0.5 ml PBS, and then, Rhodamine 123 dye was used for cell staining. Four hundred microliters of dye stock solution was mixed with 100 μl of cell suspension and kept in a dark place at room temperature for 10 min. Finally, bacterial cell viability was measured by flow cytometry (BD FACSCalibur). A total number of 100,000 cells were assessed for each sample [31].

Statistical Analysis
All experiments were carried out in triplicates. Obtained results were analyzed by one-way ANOVA and Tukey's multiple comparisons tests using GraphPad prism 8.0.2. The significance level of P-value < 0.05 was considered for all tests.

Isolation and Screening of Calcium Carbonate Crystal-Producing Bacterial Strains
The first screening was conducted to select the ureaseproducing bacterial strains. This qualitative urease test was performed on Christensen's medium through observation of color changing. Urease-positive bacterial strains turned the color of the plate from yellow to pink. In contrast, some isolated strains were unable to produce urease enzyme and retained the yellow color of the medium (Fig. SI2). Finally, 44 bacterial strains were selected for further research based on their ability to produce urease enzyme. The next screening test was to check the calcium carbonate crystal-producing ability in solid and liquid mediums by urease-positive strains selected in the previous step. However, among 44 selected strains, only 21 strains could precipitate calcium F. Elmi et al. 700 carbonate in the solid medium. Also, among the above 21 strains, only 11 samples showed significant precipitation in liquid media (Fig. SI3). Finally, the morphology of the calcium carbonate sediments was observed under a light microscope (Fig. 1).

Identification of Calcium Carbonate-Producing Strains
Biochemical tests were applied for the initial identification of 11 selected strains, which results of some tests (e.g., oxidase, catalase, and Gram staining) were the same and in other tests (e.g., motility, spore staining, and indole) were different (Table SI1). Finally, according to the high amount of calcium carbonate production, six isolates, including S5, S1, S4, S7, S2, and S11, were selected for molecular identification. Furthermore, these six selected strains were identified by 16S rRNA gene sequencing. Blast results showed that S5, S1, S11, and S2 had 99% identity to Bhargavaea cecembensis, Sporosarcina pasteurii, Bacillus badius, and Lysinibacillus boronitolerance, respectively. S4 and S7 strains also had 98.82% and 97.39% identity to Bacillus sp., respectively. The 16S rRNA gene sequences were deposited at NCBI Gene Bank with accession numbers: MK420381-MK420385, and MK433256 (Table SI2). The relationship between the locally isolated bacteria and their closest bacterial species can be seen in the phylogenetic tree (Fig. 2). Phylogenetic analysis also classified all isolates into phylum Firmicutes and family Bacillaceae.

Urease Activity Assay and Amount of the Calcium Carbonate Produced
Quantitative assay of urease activity revealed that S2 and S7 strains had the highest urease activity (10.35 U/ml), and S1 strain had the lowest urease activity (3.08 U/ml), as shown in Fig. 3b. Also, the maximum and minimum values of calcium carbonate were determined for S5 and S1 strains (410 and 143 mg/100 ml), respectively (Fig. 3). Finally, the S5 strain was selected for further study because the ability of calcium carbonate precipitation has not yet been reported for this strain and it also has high urease activity and a high amount of calcium carbonate production.

Detection of Nanocalcite by XRD, TEM, and SEM-EDX
XRD pattern showed that the selected isolate could form calcite and vaterite polymorphs (Fig. 4). The shape of crystals was observed by SEM analysis. As depicted in Fig. 5a, vaterite and calcite crystals had irregular and nanoparticle morphologies (respectively), that irregular crystals had a smooth surface, and calcite nanoparticles were aggregated together. Crystal sizes ranged between 25 and 42 nm (Fig. 5c). Furthermore, their elements were detected by the EDX method. The results showed that three constituent elements of sediments were oxygen, calcium, and carbon. Therefore, it can be concluded that produced sediments were calcium carbonate (Fig. 5d). Moreover, the TEM image (Fig. 5b) revealed that the formed crystals were aggregated nanoparticles and irregularly shaped at the nanometer scale. Also, TEM images showed abundant nanocrystalline calcite.

Growth of Isolated Strain in Extreme Conditions
The isolated strain showed the ability to grow and adapt in the temperature range of 20 to 50 °C (Fig. 6a). Its growth rate decreased with increasing temperature, while the growth stopped at temperatures above 50 °C. The highest bacterial growth was obtained when the culture was conducted at 30 °C.
Next, the tolerance of this strain to acid and alkaline stress was investigated (pH 1 to 9). As shown in Fig. 6b, the S5 strain was capable of growing in highly acidic and alkaline conditions, but optimal growth was observed at PH = 7. Finally, the stimulating or inhibitory effects of salinity stress on strain growth were studied. According to the obtained results (Fig. 6C), the S5 strain could grow in the presence of various concentrations of sodium chloride (2 to 25% w/v), but the best growth was viewed in a medium without NaCl. Therefore, it can be concluded that the selected strain is a polyextremophile bacteria with a high tolerance to harsh environmental conditions.

Flow Cytometric Measurement of Cell Viability After Desiccation
The effect of desiccation stress on cell viability was investigated by using the flow cytometry technique after 28 days of desiccation. The results obtained showed that 96% of the cells were alive, and only 4% of them were eliminated. Figure 7 is a comparison of cells numbers between the desiccation test sample and positive control stained with rhodamine. It represented that the fluorescence intensity of peak b (desiccated sample) was within the 10-100 range, which was the same as the positive control (peak a), but peak b was slightly shorter than peak a, which indicates a minor reduction in the number of living cells. Therefore, it can be said that the S5 strain can withstand drying stress and survive well under such conditions.

Discussion
MICP is a phenomenon that occurs naturally everywhere, such as aquatic and terrestrial environments [2]. This ecofriendly process has received much attention in recent years [32]. Therefore, the current study explored the novel ureaseproducing soil bacteria for the application of MICP. Among the selected strains, this is the first finding of the ability of Bhargavaea cecembensis to produce nano-sized calcium carbonate crystals. So far, there has been no report on its MICP potential in bio-cementation studies. Also, our results showed that it has a high level of urease activity (8.16 U/ ml) and it is similar to the urease activity of Sporosarcina pasteurii, for which Yang et al. reported a range of 7-11 U/ ml at different temperatures and pH [33]. Therefore, this strain was chosen for the next steps of our research. This novel bacterium belongs to the Firmicutes and it was first isolated from deep-sea sediments by Manorama et al. in 2009 and reported as a new genus [34]. Biochemical tests indicated that the strain, S5 is a Gram-positive, rod-shaped, non-motile, oxidase-positive, catalase-positive, and nonspore-forming bacterium, and our findings are in agreement with the results reported by Hammes et al. [34]. The use of indigenous bacteria has some advantages, such that they adapt well to native conditions and these bacteria are less likely to become pathogenic compared to exogenous bacteria under the influence of stressful situations [35]. Thus, this study can open up new horizons in the use of non-pathogenic ureolytic species isolated from Iran, Khuzestan, and they can be considered as potential local candidates for urease enzyme production and bio-cementation.
In this paper, the mineralogical analysis demonstrated that calcite and vaterite were the dominant forms of crystals produced by the strain, S5. This is in agreement with the   [38]. An important point about calcifying bacteria is their resistance to harsh environmental conditions, which has not been received due attention because most applications of MICP are used in extreme conditions for the growth of common species of bacteria. For example, concrete lacks the essential nutrients for the growth and activity of microorganisms [18] or desert regions generally lack sufficient water and may be too hot or too cold. For this reason, our next objective was to study the effect of extreme conditions on the growth of the selected strain. Bacterial growth was observed at high temperatures up to 50 °C, high salt concentrations (15-20% w/v NaCl), and alkaline pH of 9-11. The results also showed that the S5 strain had 96% viability after drought stress. Given the similarities found between the results obtained from our study and the findings of previous studies about some polyextremophiles (Table SI3) [39,40], it can be concluded that our strain is a polyextremophile bacterium with a high resistance to adverse conditions, such as high temperature, alkaline pH, salinity, and dryness. Extremophilic microorganisms are an excellent source of new biomolecules and enzymes, which are useful for different applications in biotechnology and industrial processes [41]. Thus, the polyextremophile calcifying strain isolated in the present study can be a suitable candidate for increasing the strength and stability of concrete, dust control, and other engineering projects. It is necessary to conduct more research and investigations on the application of economical and eco-friendly biotechnological methods in soil stabilization and cementation technology as an alternative to other traditional methods.

Conclusions
In summary, the present study is the first finding of the ability of Bhargavaea cecembensis to precipitate nanocrystalline calcium carbonate among the researches that have been done so far. This bacterium was first reported in 2009 as a new genus. This strain is a polyextremophile calcifying bacterium which can be introduced as a suitable candidate for bio-cementation under difficult and adverse conditions and also can be a good choice for soil stabilization, concrete crack repair, and other eco-friendly engineering activities.
Author Contribution F.E. conducted the experiments, carried out data analysis, and wrote the first draft. Z.E. and G.E. designed and supervised the research. Z.E. also reviewed the manuscript and assisted with data analysis. All authors read and approved the final manuscript.
Funding This study was supported by a grant from the University of Isfahan, Iran.
Data Availability All data generated during this study are included in this published article. Fig. 6 Growth patterns of S5 strain in different temperatures (a), pHs (b), and NaCl concentrations (c). The S5 strain was inoculated in N.B medium and incubated at different temperatures with 150 rpm shaking for 10 days. The effects of NaCl and pH were investigated at the optimum temperature