# The Efficiency of Calcium Oxide on Microbial Self-Healing Activity in Alkali-Activated Slag (AAS)
## 2 Experimental Program
### AAS Mortar Manufacture  
Alkaline solution blending was the first step in AAS manufacture. After blending the solutions, a certain water content was added according to the design steps. The design procedure was conducted by solving the equations of concrete absolute volume along with the equations of alkali dosage (Na\({}_{2}\)O%), modulus silicates (Ms), and liquid-to-binder ratio (L/b) simultaneously (Equations (1)-(4)) [29]. The ratio of slag to fine aggregate was kept at 1:3 based on the recommendations of the Egyptian Code of Practice (ECP 203) [30]. During the AAS mixing, solid constituents, including slag, calcium oxide powder, and fine aggregate, were perfectly mixed in a 5-L capacity Hobart mixer for 3 min (Figure 2a). After that, the liquid solutions were carefully added to the solid mixture for a further 2 min. For the microbial-induced AAS mixtures, _Bacillus subtilis_ culture at a concentration of 10\({}^{5}\) cells/mL was gently added to the AAS mix in the form of the calculated water content as per volume. The mixing process continued for 2 min. Before the mixing process, \begin{table}
\begin{tabular}{c c} \hline \hline
**Characteristics of _Bacillus subtilis_** & **Value** \\ \hline Growth medium & 3 \\ Incubation time & 24 h \\ Subculture & 30 days \\ Gram stain & Positive \\ shape & Rod \\ Oxygen demand & Facultative \\ \hline \hline \end{tabular}
\end{table} 
Table 4: _Bacillus subtilis_ pure culture characteristics. Figure 1: _Bacillus subtilis_ culture. metallic molds were oiled and prepared for mortar pouring. Demoulding took place after 24 h of the AAS manufacturing process. A water curing regime at room temperature (21 \({}^{\circ}\)C \(\pm\) 2) was applied for AAS specimens incorporating microbes for 7 and 28 days to provide an adequate environment for the microbes' survival (Figure 2b). To assess the bacteria's efficiency in healing cracks, the pre-cracking phase was considered for some specimens. Pre-cracking was carried out by subjecting AAS specimens to a compression load of 85% of their ultimate load capacities. For evaluating the crack healing progress, photo documentation was performed before and after subjecting the specimens to water curing to motivate the healing process. \[\frac{\text{Slag}}{\text{SG}_{\text{slag}}}+\frac{\text{Na}_{\text{2}}\text{ SiO}_{3}}{\text{SG}_{\text{Na}_{\text{2}}\text{SiO}_{3}}}+\frac{\text{NaOH}}{\text{SG}_{ \text{NaOH}}}+\frac{\text{H}_{\text{2}}\text{O}}{\text{SG}_{\text{H}\text{O}}} =1000\text{ L}-\frac{\text{CA}}{\text{SG}_{\text{CA}}}-\frac{\text{FA}}{\text{SG}_ {\text{FA}}} \tag{1}\]  
where slag is the weight of slag content in kg, \(\text{SG}_{\text{X}}\) indicates the specific gravity of the corresponding material, x, \(\text{Na}_{\text{2}}\text{SiO}_{3}\) shows the weight of sodium silicates in kg, NaOH is the weight of sodium hydroxide in kg, H\({}_{\text{2}}\)O represents the weight of water in kg, CA is the coarse aggregates weight in kg, and FA shows fine aggregates weight in kg. \[\text{Na}_{\text{2}}\text{O}\%=\frac{\text{Na}_{\text{2}}\text{O}\%\text{ in NaOH}+\text{Na}_{\text{2}}\text{O}\%\text{ in Na}_{\text{2}}\text{SiO}_{3}}{\text{Mass of slag}} \tag{2}\]  
\[\text{Ms}=\frac{\text{SiO}_{\text{2}}\%\text{ in Na}_{\text{2}}\text{SiO}_{3}}{\text{Na}_{\text{2}}\text{O}\%\text{ in Na}_{\text{2}}\text{SiO}_{3}+\text{Na}_{\text{2}}\text{O}\text{ in NaOH}} \tag{3}\]  
\[\text{L}/\text{S}=\frac{\text{Water in all solutions}}{\text{Slag}+\text{Na}_{\text{2}} \text{O}\%\text{ solids}+\text{SiO}_{\text{2}}\text{ solids}} \tag{4}\]  
where \(\text{Na}_{\text{2}}\text{O}\%\) in NaOH and \(\text{Na}_{\text{2}}\text{O}\%\) in \(\text{Na}_{\text{2}}\text{SiO}_{3}\) are the percentages of sodium oxide solids in the sodium hydroxide and sodium metasilicate solutions, respectively, \(\text{SiO}_{\text{2}}\%\) in \(\text{Na}_{\text{2}}\text{SiO}_{3}\) represents the percentage of silicon oxide solids in the sodium metasilicate solution. Water in all solutions indicates the percentage of water in the mixture.