## 3 Results and Discussion
### Effect of Microplastics at Different Biofilm Developmental Stages  
The time-dependent biofilm formation of PAO1 was observed in an empty 24-well plate before testing the growth of the PAO1 biofilm formed on the microplastic surface. The amount of biofilm was not large at 1 or 2 h, but it significantly increased after 4 h (Figure 4a). These observations indicated that the initial attachment and aggregation phase proceeded for about 2 h, followed by the biofilm formation and growth phase between 2 h and 6 h.  
The time-dependent biofilm formation of PAO1 was also assessed by performing biofilm tests in the presence of PP, PE, PS, and PTFE (Figure 4). To compare biofilm formation on two substrates (i.e., the well surface and microplastic surface) with different surface areas, the number of biofilms formed per surface area was presented. During the first 1-2 h (initial attachment and aggregation phase), more microorganisms were attached  
Figure 4: (**a**) Biofilm formation on the well surface. (**b**) Biofilm formation on the microplastic surface. Error bars were defined as standard deviations (n = 6, biological replicates).  
Figure 3: (**a**) Images of microplastics in water. (**b**) The possible mechanism of biofilm detachment from the microplastic surface.  
to the microplastic surface (4.30-8.92 mm\({}^{-2}\)) than to the well plate (2.15-3.49 mm\({}^{-2}\)). The 24-well plate used in this study was made of PS, and its surface hydrophilicity was increased by changing electrostatic characteristics through surface treatment, making hydrophobic microorganisms less able to attach to it. PE (8.36-8.92 mm\({}^{-2}\)) had a larger amount of microorganisms attached to its surface than PP (5.37-5.77 mm\({}^{-2}\)) (Figure 4b), which could be due to the higher hydrophobicity of PE. However, among the four microplastics, PTFE, which had the highest hydrophobicity, allowed the lowest attachment of microorganisms on its surface. This is presumably due to the maximum density of the PTFE, which caused it to sink to the bottom and frequently collide with the surface of the well plate, as explained in Section 3.2.  
Unlike the initial attachment and aggregation phase (1-2 h), the biofilm on the well surface increased exponentially during the biofilm formation and growth phase (2-6 h), whereas it did not significantly increase on the microplastic surface (Figure 4). This could be explained by the collision between the microplastics and the well plate. The entire surface area of microplastics was exposed to the collisions, whereas only a specific part of the well plate, the upper or lower part, was exposed to the collisions with microplastics. This is presumed to be the cause of the failure of the biofilm to grow exponentially on the microplastic surface as on the well plate surface. A previous study that investigated bacteria-plastic interactions with various types of flat plastic surfaces reported that the surface properties of plastics, especially surface hardness, play an important role in the adhesion of bacteria [34]. However, as in the current study, when the plastic particles move, it can be seen that the behavior in water according to the density of plastic particles has a decisive effect on biofilm formation.