**3.6 Microbial precipitation CaCO3**

As has already been mentioned, the application of bacteria in concrete for the purpose of self-healing in case of cracks is not impossible. There have been many studies that prove their effectiveness in healing cracks. Bacteria that can withstand the extreme environment of concrete (high voltages and intense alkalinity) are those of the genus Bacillus (Gram-positive bacteria). Species of this genus have the potential to form endospores, forms resistant to non-bacterium-friendly environments. When the endospores are found in appropriate conditions, they are activated (a process also known as eblastosis) and begin to develop. Bio-precipitation of a mineral by a microorganism occurs when it is found in an environment with suitable nutrient material. In the case of cement self-healing, the mineral chosen is CaCO3. Although the most suitable bacterium for this work is some species from the genus Bacillus, this study looked at two other types of bacteria, namely *Escherichia coli* (Gram negative) and *Staphylococcus aureus* (Gram positive). Thus, the possibility of submersion of CaCO3 salt from a Gram-negative and a Gram-positive bacterium was studied, with the main emphasis on *Staphylococcus aureus*. The reason this was done is because *St. aureus* has a cell structure similar to that of Bacillus, although it does not have the ability to form endospores. It should be stressed that the bacteria studied in this work were pathogenic, and all safety rules were followed during their use. As mentioned in the above section, CaCO3 precipitation is a two-way chemical process controlled mainly by four factors, first calcium concentration, second concentration of dissolved inorganic carbonate ions (DIC), third the pH, and fourth availability of nucleation centers.

There are three mechanisms associated with bio-precipitation, and in this work, the mechanism of urea breakdown (hydrolysis of urea, HU) was studied through the enzyme urease, a course easily manageable and controlled. The general reaction is as follows:

$$\text{CaO}\_3^{2-} + \text{Ca}^{2+} \rightarrow \text{CaCO}\_3 \tag{2}$$

The bacterium plays the role of the nuclearization center with the following mechanism:

$$\text{Ca}^{2+} + \text{Cell} \rightarrow \text{Cell} - \text{Ca}^{2+} \tag{3}$$

$$\text{Cell} - \text{Ca}^{2+} + \text{CO}\_3^{2-} \rightarrow \text{Cell} - \text{CaCO}\_3 \downarrow \tag{4}$$

The exponential growth phase is the ideal phase to study any cellular function.

### *3.6.1 Precipitation CaCO3 from bacteria*

### *3.6.1.1 CaCO3 precipitation rating study by Staphylococcus aureus*

In order to investigate the possibility of precipitation of CaCO3 from *St. Aureus*, four microorganism solutions with a different amount of CaCl2.2H2O and urea were prepared. In this way, the ability of this bacterium to precipitate salt at different nutrient concentrations has been qualitatively studied. **Table 6** gives the composition of *St. aureus* solutions with different concentration of CaCl2.2H2O and urea.

For the preparation of the above solutions, a quantity of microorganism solution was isolated and the corresponding quantity of CaCl2.2H2O and urea was added


**Table 6.**

*Composition of St. aureus solutions with different concentrations of CaCl2.2H2O and urea.*

each time. The solutions were incubated at 37°C at 100 rpm. After 72 h, in these conditions, the solutions were removed and compared. The solution of the microorganism containing the most CaCl2.2H2O and urea showed the greatest turbidity of the other solutions with apparent precipitation of CaCO3. There is a marked change in the turbidity of the solutions, as distinguished from the images above. The change in clarity between the nutrient solution, LB and the solution after the development of the microorganism, certifies that the microorganism has been properly incubated to study any of its metabolic processes. An apparent precipitation of sediment is observed between the 5th solution and the microorganism solution (**Figure 14C**). *St. aureus* solution becomes cloudier while CaCO3 submersion certifies its ability to break down urea and lead to precipitation of the mineral. In order to characterize more fully the precipitation of CaCO3, a kinetic study is carried out by the microorganism in solutions of (a) in nutrient material LB in the presence of CaCl2.2H2O and urea and (b) in agar medium (Petri dish) in the presence of CaCl2.2H2O and urea. Taking samples at regular intervals, and through infrared spectroscopy (FT-IR), the existence of the mineral is studied in two different environments. The following describes the composition of microorganism solutions in LB nutrient solution and

### **Figure 14.**

*Change in clarity of St. aureus solution. (A) LB solution, (B) microorganism solution after incubation in LB, and (C) precipitation of CaCO3 from the 5th solution of the bacterium. Change in the turbidity of the E-coli crop: (D) LB solution, (E) microorganism solution after incubation in LB, and (F) precipitation of CaCO3.*

### *Self-Healing of Concrete through Ceramic Nanocontainers Loaded with Corrosion Inhibitors… DOI: http://dx.doi.org/10.5772/intechopen.93514*

agar nutrient medium for the CaCO3 precipitation kinetic study. Maximum concentrations of CaCl2.2H2O and urea were selected from CaCO3 precipitation quality control, as in these quantities the mineral was more evident.

In this particular case, however, *St. Aureus*, which develops, is placed in the stirring incubator for approximately 24 days in certain conditions (37 °C, 100 rpm). At regular intervals, a sample is isolated, centrifuged, and sterilized to measure the FT-IR spectrum for the purpose of certifying the presence of CaCO3.

In this particular case, however, ο *St. aureus* is left in the incubation oven for about 21 days under certain conditions. (37 °C). At regular intervals a sample is isolated, centrifuged, sterilized and measured the FT-IR spectrum for the purpose of certifying the presence of CaCO3. The environment in which the mineral develops is quite complex, with many different compounds, which come from the nutrient and microorganism. These compounds show peaks in IR spectra. Therefore, in order to verify the existence of CaCO3 in the cases where the study is carried out, a comparison of the range of CaCO3 with the spectrum CaCO3 LB nutrient solution (**Figure 15A**), in order to identify the tops of the mineral in the nutrient microorganism. Then, after determining the carbonic ion peaks in the IR spectrum, each kinetic study spectrum is included in addition to the spectra of the isolated samples, the CaCO3 LB and the spectrum of the microorganism. In this way, it is possible to identify the peaks corresponding to the CaCO3 in any case but also the peaks corresponding to the microorganism. The results of this study are listed below.

Since CaCO3 is a crystalline salt, the vibrations that occur in an IR spectrum correspond to the bonds of CO3 2−. The carbonic ion peaks in the IR spectrum are as follows: a strong wide peak in the 1530–1320 cm−1 range, medium intensity peaks at 1160 cm−1 and in the 890–800 cm−1 range, and a peak in the 745–670 cm−1 range [5]. Based on the above and the IR spectrum of **Figure 15A**, we observe that CaCO3 in LB solution shows peaks at 712, 873, and 1409 cm−1. The top at 1638 cm−1 can be attributed to any of the components of the nutrient medium LB. Finally, the peak shown in the range 3000–3500 cm−1 is attributed to the –OH of the aqueous solution. An indicative range of IR bacterium is shown in **Figure 15B**. Based on

### **Figure 15.**

*(A) IR spectrum of the crystalline CaCO3 and CaCO3 in the nutrient medium LB, (B) spectrum IR St. aureus in nutrient medium LB, (C) IR spectrum of CaCO3 precipitation kinetic study from St. aureus to nutrient LB, and (D) IR spectrum of CaCO3 kinetic precipitation study from St. aureus in the nutrient agar medium in a dish petri.*

the range of the bacterium, we can assign some peaks to the various components that make up the microorganism. The peak at 1018 cm−1 is attributed to polysaccharides compounds, the peak at 1260 cm−1 at the asymmetric vibration of the PO2 cm−1 bond, at 3274 cm−1 we observe the vibration of N-H, while at 2838 cm−1 we observe the symmetrical vibration of the CH2 bond [5]. The IR spectrum of the CaCO3 kinetic precipitation study from *St. aureus* to the nutrient medium LB is given below. By comparing the spectra of the samples isolated each time, the spectra of the microorganism and CaCO3 in LB draw the following conclusions. From the 3rd day of incubation of the microorganism with CaCl2.2H2O and urea, precipitation CaCO3 is observed. This is certified from the top at 1468 cm−1, which as mentioned above belongs to the display area 1530–1320 cm−1 for carbonate ions. The new peak at 780 cm−1 is also attributed to carbonate ions. Although this peak does not correspond to any area of appearance of carbonate ions, it can be attributed to the range 745–670 cm−1 but as shifted. The presence of carbonate ions is observed in the kinetic study for all periods of time when the sample is isolated. The remaining peaks showing the sample spectra can be attributed to compounds of the microorganism and nutrient material. Finally, with this comparative study of the various IR spectroscopy, it appears that *St. aureus* is capable of precipitating CaCO3 in LB nutrient solution when dissolved in sufficient CaCl2.2H2O and urea. Due to the ureolytic action of the microorganism, carbonate ions are released, and in a calcium-rich environment there is precipitation of the mineral. Below is the IR spectrum of the kinetic precipitation study CaCO3 from *St. aureus* to the nutrient in Petri dish. The comparative precipitation study of CaCO3 by St. aureus in the nutrient medium, in a Petri dish, observed the carbonic peaks corresponding to carbonate ions in the isolated samples. Thus, according to the above range, the IR peak at 1638 cm−1 is attributed to nutrients and the peak to 1031 cm−1 to the polysaccharides of the microorganism. The peak attributed to carbonate ions is in the range 1399–1457 cm−1, as it is included in the range 1530–1320 cm−1 where these ions appear. We can conclude, for example, that *St. aureus* has the ability to precipitate CaCO3 in a Petri dish (**Tables 7** and **8**).
