ZnO Nanoparticles Affect <i>Bacillus subtilis</i> Cell Growth and Biofilm Formation

<div><p>Zinc oxide nanoparticles (ZnO NPs) are an important antimicrobial additive in many industrial applications. However, mass-produced ZnO NPs are ultimately disposed of in the environment, which can threaten soil-dwelling microorganisms that play important roles in biodegradation, nutrient recycling, plant protection, and ecological balance. This study sought to understand how ZnO NPs affect <i>Bacillus subtilis</i>, a plant-beneficial bacterium ubiquitously found in soil. The impact of ZnO NPs on <i>B</i>. <i>subtilis</i> growth, FtsZ ring formation, cytosolic protein activity, and biofilm formation were assessed, and our results show that <i>B</i>. <i>subtilis</i> growth is inhibited by high concentrations of ZnO NPs (≥ 50 ppm), with cells exhibiting a prolonged lag phase and delayed medial FtsZ ring formation. RedoxSensor and P<i>_hag</i>-GFP fluorescence data further show that at ZnO-NP concentrations above 50 ppm, <i>B</i>. <i>subtilis</i> reductase activity, membrane stability, and protein expression all decrease. SDS-PAGE Stains-All staining results and FT-IR data further demonstrate that ZnO NPs negatively affect exopolysaccharide production. Moreover, it was found that <i>B</i>. <i>subtilis</i> biofilm surface structures became smooth under ZnO-NP concentrations of only 5–10 ppm, with concentrations ≤ 25 ppm significantly reducing biofilm formation activity. XANES and EXAFS spectra analysis further confirmed the presence of ZnO in co-cultured <i>B</i>. <i>subtilis</i> cells, which suggests penetration of cell membranes by either ZnO NPs or toxic Zn⁺ ions from ionized ZnO NPs, the latter of which may be deionized to ZnO within bacterial cells. Together, these results demonstrate that ZnO NPs can affect <i>B</i>. <i>subtilis</i> viability through the inhibition of cell growth, cytosolic protein expression, and biofilm formation, and suggest that future ZnO-NP waste management strategies would do well to mitigate the potential environmental impact engendered by the disposal of these nanoparticles.</p>