## 3 Test Plan for Enhanced Immobilization of Bacteria through NPM
### Test Program Outline for Engineering Property Evaluation
#### 3.2.3 Strength and Resistance to Sulfate Attack  
The specimen used for the evaluation of the strength of the bio-coating material (repair with mixed bacteria) was prepared as described in ASTM C 39. After curing in an environment of 20 \({}^{\circ}\)C with R.H. of 60 \(\pm\) 5% for 3 days, the mold was removed and the specimen was further cured in an underwater environment at 20 \({}^{\circ}\)C for 28 days. In the case of the sulfuric acid solution immersion test, the evaluation was performed by coating the repair material on the cured concrete specimen with a diameter of 100 mm and a height of 200 mm. The repair material was coated on the concrete with a thickness of 2.5 mm on the 28 th day in consideration of the economic aspect and the efficient repair of the concrete sewage structure. Specimens coated with bio-coating material were cured underwater at 20 \({}^{\circ}\)C during 28 days, and immersed in 5% sulfuric acid solution as described in ASTM C 7401. Then, with increasing immersion periods (1, 7 and 28 days), the compressive strength and mass of the specimen were evaluated based on ASTM C 39 and ASTM C 642. Three samples were prepared and the averages were used for the results. The variations in compressive strength in the sulfuric acid solution were compared considering the repair conditions and duration period.  
For the evaluation of the sulfate ion diffusion coefficient, a natural diffusion cell test was adopted. As described in ASTM C 1202, after a specimen was placed between two cells, the change in ion concentration was monitored as a function of time, and the result was derived as shown in Equation (5):  
\[D_{i}=\frac{V\Delta Q}{A\Delta T}\times\frac{L}{c_{1}-c_{2}} \tag{5}\]  
where \(V\) is the volume of the diffusion cell (m\({}^{3}\)); \(\Delta Q\) is the increase in sulfate ion concentration in the cell containing deionized water (kg/m\({}^{3}\)); \(A\) is the exposed area of the sliced specimen (m\({}^{2}\)); \(\Delta T\) is the elapsed time after starting the test (s); \(L\) is the thickness of the sliced specimen (m); \(c_{1}\) is the concentration of the sulfate ion solution (kg/m\({}^{3}\)); and \(c_{2}\) is the average concentration of the cell containing deionized water (kg/m\({}^{3}\)).  
The diffusion coefficient was evaluated by retaining the specimen for 56 days, after which the concentration became almost constant under steady condition. The volume of the cell containing the solution with sulfate ions (SO\({}_{4}\)\({}^{2-}\)) and the deionized water was 0.001 m\({}^{3}\), and the concentration of sulfate ion in the solution with sulfate ions was set to 120 ppm (1.24 mol/m\({}^{3}\)). The thickness of the repair material slice was 5 mm, and the area exposed to deionized water and the sulfate ion solution was 70 mm \(\times\) 70 mm. In addition, the change in the sulfate ion concentration in the cell was recorded using an ion meter with which data can be checked in real time. Figure 4 shows the specimen preparation for evaluating the resistance to the sulfate ingress of bio-coating material and photos for the test.  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Nutrients** & **Quantity** \\ \hline Yeast extract & 1.0 g/L \\ \hline Disodium succinate hexahydrate & 1.0 g/L \\ \hline Absolute ethanol & 1 mL/L \\ \hline KH\({}_{2}\)PO\({}_{4}\) & 0.5 g/L \\ \hline MgSO\({}_{4}\)\({}_{2}\)O & 0.4 g/L \\ \hline CaCl\({}_{2}\)-H\({}_{2}\)O & 0.005 g/L \\ \hline NaCl & 0.4 g/L \\ \hline Trace-element solution & 1 mL/L \\ \hline Carbon source: malic acid & Medium wt. \(\times\) 0.3\% \\ \hline \hline \end{tabular}
\end{table}
Table 3: Medium composition for culturing _Rlodobacter capsulatus_.