## 3 Results
### Effect of Ozone-Induced Free Radical Solution on Microbial Killing Mechanism and Disease Control  
3.3.1. Inhibitory Effect of Ozone-Induced Free Radical Solution on _Bacillus subtilis_  
The bactericidal rate of the ozone-induced free radical solution against _Bacillus subtilis_ escalates as the mass concentration of TRO increases. At a concentration of 0.3 mg/L, the sterilization rate stood at 51.53%, rising to 87.63% at 0.5 mg/L, and exceeding 99.9% at 1.2 mg/L. Table 1 demonstrates the sterilization rate is directly proportional to the number of free radicals interacting with the bacterial suspension. The sterilization rate of an ozone-induced free radical solution on _Bacillus subtilis_ reached 85.10% at 1 min and 97.76% at 10 min, with the growth rate of the sterilization rate decelerating after 5 min. Generally, Table 2 demonstrated that a longer exposure time results in a more effective bactericidal outcome. \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Solution Concentration (mg/L)**} & \multicolumn{2}{c}{**Mean Number of Colonies (cfu/mL)**} \\ \cline{2-3}  & _Bacillus subtilis_ & **Germicidal Ratio (\%)** \\ \hline Control group & \(2.95\times 10^{5}\) & 0 \\
0.3 & \(1.43\times 10^{5}\) & 51.53 \\
0.5 & \(3.65\times 10^{4}\) & 87.63 \\
0.8 & \(1.8\times 10^{4}\) & 93.9 \\
1.2 & 15 & 99.99 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The bactericidal effect of the free radical solution at different concentrations. \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Treatment Time (min)**} & \multicolumn{2}{c}{**Mean Number of Colonies (cfu/mL)**} \\ \cline{2-3}  & _Bacillus subtilis_ & **Germicidal Ratio (\%)** \\ \hline Control group & \(2.45\times 10^{4}\) & 0 \\
1 & \(3.65\times 10^{4}\) & 85.1 \\
3 & \(2.2\times 10^{4}\) & 91.02 \\
5 & \(1.95\times 10^{4}\) & 92.04 \\
10 & \(5.5\times 10^{3}\) & 97.76 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Effect of treatment time on sterilization by free radical solution. Figure 13: UV absorption spectrum after DPBF reaction. 3.2 Analysis of the Effect of the Ozone-Induced Free Radical Solution on the Structure of _Bacillus subtilis_  
The impact of the solution on the external structure of _Bacillus subtilis_ was assessed using scanning electron microscopy. Figure 14a presents an electron microscope image prior to solution treatment, depicting the bacteria as straight, rod-shaped, with blunt ends, and uniformly sized. Figure 14b displays the electron microscope image following treatment with a 0.3 mg/L solution, where the cells' external structure exhibits wrinkles and depressions, and the cells appear shrunken without rupture--evidence of the reaction between the ozone-induced free radical solution and the bacteria. Figure 14c shows the electron microscope image post-treatment with a 0.5 mg/L solution, revealing significant cracks, slimes, and pits on the bacterial surface, signifying that a higher mass concentration of TRO of the ozone-induced free radical solution exacerbates cell structure damage. Damage to the internal structure of _Bacillus subtilis_ caused by the solution was evaluated using transmission electron microscopy. Figure 15a depicts the transmission electron microscopy image of the bacteria prior to the solution treatment, showcasing clear visibility of the cell wall and membrane, presence of the periplasmic space, uniform cytoplasm, and visible free organelles. The cell cross-section length measured 560.93 nm (with a scale of 200 nm, applicable to subsequent descriptions). Figure 15b presents the transmission electron microscopy image after treatment with a 0.3 mg/L solution, where the periplasmic space vanished, the distinction between cell wall and membrane blurred, the cytoplasm appeared abnormal, and the cell cross-section length reduced to 548.97 nm. Figure 15c illustrates the transmission electron microscopy image post-0.5 mg/L solution treatment, revealing a thinner cell wall, emergence of air chambers and gaps between the cell walls, and a cell section length of 508.58 nm.