Flow cytometry analysis of <i>P</i>_<i>hag</i><i>-GFP</i> at various concentrations of ZnO NPs.

<p>Wild-type bacteria were grown at different ZnO-NP concentrations for 3 h. <b>A</b>: PBS, <b>B</b>: unstained cells, <b>C</b>: 0 ppm, <b>D</b>: 10 ppm, <b>E</b>: 25 ppm, <b>F</b>: 50 ppm, and <b>G</b>: 100 ppm. The X axis indicates GFP fluorescence intensity (arbitrary units: au), and the Y axis indicates cell counts.</p

XANES and EXAFS spectra for <i>B</i>. <i>subtilis</i> cells treated with ZnO NPs.

<p><b>A.</b> ZnO K-edge XANES spectra of silver standards and <i>B</i>. <i>subtilis</i> cells treated with 100 ppm of ZnO-NPs. <b>B.</b> ZnO K-edge EXAFS spectra of ZnO standards and <i>B</i>. <i>subtilis</i> cells treated with 100 ppm of ZnO-NPs. The best-fitting EXAFS spectra are indicated by the colored symbol lines. (Zn-Zn standard: red; ZnO standard: blue; <i>Bacillus subtilis</i> cells treated with 100 ppm of ZnO-NPs: black).</p

ZnO NPs affect biofilm formation.

<p><b>A.</b> The pellicle column depicts microtiter wells (6-well plate) in which cells were grown in biofilm medium with various concentrations of ZnO NPs at 25°C for 3 days (scale bar: 2 cm). Bacterial wild-type (3610) and mutant strains are indicated as follows: <i>sinR</i> (DS92), <i>epsA-O</i> (DS696), <i>sfp</i> (DS3629), <i>tasA</i> (DS3630), and <i>sinR epsA-O</i> (HS222). <b>B.</b> Images of a 12-well microtiter dish containing ethanol-precipitated supernatant from the indicated strain, following treatment with different concentrations of ZnO NPs. <b>C</b>. The supernatants of the indicated strains were treated with proteinase K, DNase, and RNase, precipitated with ethanol, and resolved through SDS-PAGE on a 12% gel, after which staining with Stains-All was performed. <b>D.</b> FT-IR spectra analysis of EPS from <i>Bacillus</i> cells treated with ZnO NPs. Wild type bacteria were grown at ZnO NP concentrations of 0, 5, 10, 25, and 50 ppm, and untreated <i>eps</i> mutant cells were grown to serve as a negative control.</p

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></div

<i>P</i>_<i>hag</i><i>-GFP</i> at varying concentrations of ZnO NPs.

<p>Fluorescent micrographs of <i>B</i>. <i>subtilis</i> cells show the expression of <i>P</i>_{<b><i>hag</i></b>}<i>-GFP</i> after cultivation with ZnO-NP concentrations of 0, 5, 10, 25, 50, and 100 ppm for 3 h. GFP reporter expression presents a false green color. DAPI presents a false blue color. Scale bar: 10 μm.</p

Synthesis and morphological analysis of ZnO NPs.

<p><b>A</b>. Scanning electron microscope image of ZnO NPs used in this study; white bar: 100 nm. <b>B</b>. Size distribution of ZnO NPs. <b>C.</b> X-ray diffraction patterns of ZnO NPs synthesized by the sol-gel method.</p

Localization of FtsZ in wild-type cells grown under concentrations of 0, 50, and 100 ppm ZnO NPs in LB at 37°C.

<p>FtsZ was stained green; cell membranes were stained red; and DNA was stained blue. The closed yellow arrows indicate medial FtsZ rings.</p

Flow cytometry and fluorescent micrograph analysis of RedoxSensor activity in <i>B</i>. <i>subtilis</i>.

<p>Wild-type bacteria were grown for 3 hrs at ZnO-NP concentrations of 0 ppm, 10 ppm, 25 ppm, 50 ppm, 100 ppm. Unstained samples and PBS buffer alone were used as controls. The X axis indicates RedoxSensor or PI fluorescence intensity (arbitrary units: au), and the Y axis indicates cell counts. <b>A.</b> Flow cytometry analysis of Redoxsensor activity, and <b>B.</b> Flow cytometry analysis of PI activity. <b>C.</b> Fluorescent micrographs of <i>B</i>. <i>subtilis</i> cells indicate RedoxSensor activity or PI fluorescence after incubation with different concentrations of ZnO-NPs for 3 h. Redoxsensor activity presents a false green color. PI presents a false red color. Scale bar: 10 μm.</p

Effects of various ZnO-NP concentrations on the growth of <i>Bacillus subtilis</i>.

<p><b>A.</b> Growth analysis curves were measured by monitoring the optical density (OD) at 600 nm. <b>B.</b> Antibacterial activity of ZnO NPs on <i>B</i>. <i>subtilis</i> cells; ZnO-NP concentrations are shown as -￭-: 0 ppm, -●-:5 ppm -▲-: 10 ppm, -▼-: 25 ppm, -◆-: 50 ppm, -◀-: 100 ppm, and -▶-: 200 ppm.</p

Effects of various Ag-NP concentrations on <i>B</i>. <i>subtilis</i> growth.

<p>(A) Growth analysis curves of <i>B</i>. <i>subtilis</i> in rich media treated with Ag NPs, measured by monitoring OD₆₀₀ values. (B) Growth analysis curves of <i>B</i>. <i>subtilis</i> in minimal media treated with Ag NPs. Ag-NP concentrations are shown as -▢-: 0 ppm; -▷-: 0.1 ppm; -○-: 1 ppm; -△-: 5 ppm; -▽-: 10 ppm; -◇-: 25 ppm; and -◅-: 50 ppm. (C) Ag-NP antibacterial activity against <i>B</i>. <i>subtilis</i> cells.</p