## 3 Results and Discussion
### Biofilm Formation on Microplastic Surfaces  
_P. aeruginosa_ PAO1 was incubated in a 24-well plate with each microplastic under shaking conditions, and the amount of biofilm formation on the surface of the well plate and the microplastic was measured. When comparing the amount of biofilm formed on the surface of the well plate, the wells injected with PS and PTFE (OD550: 1.38-1.88) produced more than the wells injected with PP and PE (OD550: 0.97-1.27) (Figure 2a). Conversely, the biofilms on the microplastic surfaces were observed to form more PP and PE (OD550: 0.39-0.40) than on PS and PTFE (OD550: 0.26-0.28) (Figure 2b). As stated in Section 3.1, the higher the hydrophobicity of the plastic, the better the adhesion of microorganisms and biofilm formation. However, in this study, although PTFE had the highest hydrophobicity, it had the least amount of biofilm formed. Also, in the case of other plastics, there was no significant correlation between the amount of biofilm formation and the degree of hydrophobicity of the plastics. This experiment revealed that biofilm formation may be influenced by factors other than the hydrophobicity of microplastics.  
The reduced biofilm formation on the PS and PTFE surfaces could be due to the relatively high densities of the two plastics. Since PS and PTFE have a higher density than water (Table 1), they sink to the bottom of the well plate (Figure 3a). During the incubation period, the microplastic moves and collides with the surface of the well plate. During this process, PS and PTFE collide with the bottom and wall surface of the well. However, PP and PE, which have similar densities to water and float on the culture medium (Figure 3a), collide only with the wall surface, as shown in Figure 3b. As a result of the frequency of impacts, biofilms on PS and PTFE surfaces appear to be more easily peeled off. Furthermore, oxygen concentration, which is an important factor in biofilm formation and dispersion,  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline  & **PE** & **PP** & **PS** & **PTFE** \\ \hline Rq* (nm) & 116.42 & 55.09 & 119.28 & 71.24 \\ \hline \hline \end{tabular}
* Rq is one of the most commonly reported measures of surface roughness from AFM images.  
\end{table}
Table 2: Analysis of surface roughness of microplastic beads by AFM.  
Figure 2: (**a**) Biofilm formation on the well surface. Error bars were defined as standard deviations (n = 8, biological replicates). One-way ANOVA was performed and the Tukey post hoc test was conducted to compare the number of biofilms where significant differences are indicated as follows: NS, not significant \(p\) > 0.05, * \(p\) < 0.05, and *** \(p\) < 0.0001. (**b**) Biofilm formation on the microplastic surface (n = 8, biological replicates). * \(p\) < 0.01.  
may also have contributed to the reduced biofilm formation on the microplastic surfaces. Although the shaking of the well plate provides oxygen to the inside of the culture medium, oxygen concentration inside the well plate is rapidly consumed by PAO1 development. Hence, oxygen concentration at the bottom surface may be lower than the water surface in contact with oxygen [32]. Therefore, in the case of microplastics moving in a low oxygen region, such as PS and PTFE, it is assumed that biofilm does not grow effectively or is easily dispersed [33]. The biofilm formed on the surface of the PS and PTFE microplastics was low because of the influence of movement characteristics rather than surface characteristics.