## Materials and Methods
### Removal of Heavy Metals from Aqueous Solutions Using Acclimatized S. pasteurii  
In order to learn about the ability of acclimatized _S. pasteurii_ mineralizing heavy metals, and how the urea and Ca\({}^{2+}\) concentration affect the behavior of heavy metal biomineralization, a series of experiments were designed in this study. Excessive concentrations of cadmium, copper and lead have strong toxic effects on animals, plants and humans, and are easy to cause health risks and endanger the ecosystem stability. These three heavy metals are very frequently observed among heavy metal pollution issues, and are also widely studied in previous studies. Different concentrations of heavy metal (i.e., Cd\({}^{2+}\), Cu  
\begin{table}
\begin{tabular}{c c c c c} \hline Group & pH & \(T\) (\({}^{\circ}\)C) & \(t\) (h) & \(C_{\text{urea}}\) (mol/L) \\ \hline
1 & 7.0 & 30 & 0, 12, 24, 36, 48, 60, 72 & 0.32 \\
2 & 7.0 & 10, 15, 20, 25, 30, 35, 40, & 48 & 0.32 \\
3 & 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 & 30 & 48 & 0.32 \\
4 & 8.0 & 30 & 48 & 0, 0.08, 0.16, 0.32, 0.4 \\ \hline \end{tabular}
\end{table}
Table 1: Growing conditions of _S. pasteurii_.  
Figure 1: Procedure of _S. pasteurii_ acclimatization.  
and Pb\({}^{2+}\)) solutions were prepared separately, and different weights of calcium chloride were also weighed to prepare different concentrations of Ca\({}^{2+}\) to consider the effect of Ca\({}^{2+}\) concentration on the heavy metal precipitation.  
_S.p._ Cd-5, _S.p._ Cu-5 and _S.p._ Pb-5 was used for removing heavy metal Cd\({}^{2+}\), Cu\({}^{2+}\) and Pb\({}^{2+}\) from aqueous solutions, respectively. These three types of acclimatized _S. pasteurii_ were enriched in the nutrient broth at pH = 8.0 and 30 \({}^{\circ}\)C incubating for 48 h and diluted until the \(OD_{600}\) = 0.4. Then, 2 mL of the bacterial suspension was transferred into 200 mL nutrient broth with different concentrations of urea at pH = 8.0 and 30 \({}^{\circ}\)C, incubating for 48 h. Subsequently, 100 mL of the heavy metal solution was added into the above 200 mL nutrient broth with corresponding acclimatized _S. pasteurii_, and 100 mL of the Ca\({}^{2+}\) solution was also instantly added into the bacterial solution. In the reaction systems formed by the three solutions, the concentration of heavy metals varied from 50, 100, 200, 400 to 800 mg/L, the concentration of Ca\({}^{2+}\) varied from 0, 0.5, 1.0, 1.5 to 2.0 mol/L, and the concentration of urea varied from 0.5, 1.0, 1.5 to 2.0 mol/L. Two parallel experiments were performed for each group and all the reaction systems were placed in a thermostatic oscillator at 180 r/min, 30 \({}^{\circ}\)C for 48 h. The test procedure of the heavy metal biomineralization is shown in Figure 2.  
At the end of the tests, the samples were taken and centrifuged at 8000 r/min for 3 min. The supernatants were collected, and heavy metal concentrations were measured by flame atomic absorption spectrophotometry (Persee TAS-990, Beijing, China). The crystal structures of the precipitates generated in the tests were detected through X-ray diffraction spectroscopy (XRD, Bruker D8 Advance Davinci, Karlsruhe, Germany), and the morphologies were also characterized using the scanning electron microscope (SEM, Hitachi SU-70, Tokyo, Japan).