## 4 Results and Discussion
### Micro-Tests after the Addition of B. pasteurii to CG and FA
#### 4.1.3 In Situ Urea Hydrolysis Experiment  
After the production of CO3\({}^{2-}\) due to the _B. pasteurii_ hydrolysis of urea (see Section 4.1.2), additional calcium chloride solution was added for the in situ test, which was conducted at three specific time points: 12 h, 36 h and 48 h. As also observed by other researchers [49; 50], the white bioprecipitate, shown in Figure 5a, formed immediately. This precipitate was used to assess whether the mineralization had occurred in the culturing system after incubating the bacteria within urea and, subsequently, adding calcium chloride.  
Based on the results of the conductivity tests (see Figure 4), in situ urea hydrolysis tests were also carried out. In particular, Figure 5 shows the comparison between the tubes containing CG group_2, FA group_1 and Reference_0 after 12 h (Figure 5b), 36 h (Figure 5c) and 48 h (Figure 5d) of urea hydrolysis, respectively. If precipitation occurred after the addition of CaCl2, then the bacteria within the solution performed mineralization and generated CO3\({}^{2-}\)[51].  
As illustrated on the left side of Figure 5b, the addition of CaCl2 after 12 h resulted in a bioprecipitate in the tube containing CG group_2. Thus, CO3\({}^{2-}\) was present in this solution, and _B. pasteurii_ were able to perform mineralization. In the other two groups (see the tubes on the right side of Figure 5b), precipitation did not occur immediately, indicating that no CO3\({}^{2-}\) was present at this time and that the urea was not hydrolyzed by _B. pasteurii_.  
Figure 4: Effect of CG and FA environments on the conductivity of _B. pasteurii_ cultures. The bacteria were cultivated in tubes and incubated for 60 h. The experimental groups were composed of _B. pasteurii_ cultivated in basic culture medium and urea with CG and FA, respectively. Reference_0 comprised _B. pasteurii_ cultivated in basic culture medium and urea. Each set of data is displayed as the difference from the 0 h data. All tests were performed in triplicate and the vertical error bars indicate the standard deviation.  
The addition of CaCl\({}_{2}\) to the three groups after 36 h (Figure 5c) immediately produced white precipitates in the two tubes on the left side (CG group_2 and FA group_1), indicating that _B. pasteurii_ in the solution were already performing the mineralization function and secreting urease to hydrolyze the urea contained in these tubes. At this time, as in the previous case, mineralization by the bacteria did not commence in Reference_0 (the tube on the right side of Figure 5c did not show any precipitation). Finally, when CaCl\({}_{2}\) was added at 48 h (Figure 5d), bioprecipitation occurred in all tubes. Therefore, at this time, the bacteria were performing their mineralization function in all three groups.  
The previous tests demonstrate that the addition of bacteria to CG or FA does not inhibit the mineralization of the bacteria, but rather, it shortens the initiation time. Indeed, the bacteria in Reference_0 took the longest time to develop their mineralization function, whereas in CG group_2, they can perform their mineralization function earlier than in FA group_1. Moreover, the results of this macroscopic analysis are consistent with those of the microscopic urea hydrolysis tests described in Section 4.1.2.