title: ZnO Nanoparticles Affect Bacillus subtilis Cell Growth and Biofilm Formation.
abstract: 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 Bacillus subtilis, a plant-beneficial bacterium ubiquitously found in soil. The impact of ZnO NPs on B. subtilis growth, FtsZ ring formation, cytosolic protein activity, and biofilm formation were assessed, and our results show that B. subtilis 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 Phag-GFP fluorescence data further show that at ZnO-NP concentrations above 50 ppm, B. subtilis 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 B. subtilis 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 B. subtilis 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 B. subtilis 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.
authors.affiliation: Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan, Taiwan.
authors.forename: Yi-Huang
authors.initials: YH
authors.lastname: Hsueh
authors.affiliation: Graduate Institute of Biomedical Sciences, and Research Center for Bacterial Pathogenesis, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Wan-Ju
authors.initials: WJ
authors.lastname: Ke
authors.affiliation: Department of Chemical Engineering and Materials Science, Yuan Ze University, Taoyuan, Taiwan.
authors.forename: Chien-Te
authors.initials: CT
authors.lastname: Hsieh
authors.affiliation: Department of Chemical Engineering and Materials Science, Yuan Ze University, Taoyuan, Taiwan.
authors.forename: Kuen-Song
authors.initials: KS
authors.lastname: Lin
authors.affiliation: Department of Chemical Engineering and Materials Science, Yuan Ze University, Taoyuan, Taiwan.
authors.forename: Dong-Ying
authors.initials: DY
authors.lastname: Tzou
authors.affiliation: Department of Chemical Engineering and Materials Science, Yuan Ze University, Taoyuan, Taiwan.
authors.forename: Chao-Lung
authors.initials: CL
authors.lastname: Chiang
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000633:toxicity*; D001412:Bacillus subtilis / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D053758:Nanoparticles / Q000737:chemistry / Q000633:toxicity* / Q000648:ultrastructure; D010316:Particle Size; D011135:Polysaccharides, Bacterial / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D012988:Soil Microbiology; D012989:Soil Pollutants / Q000737:chemistry / Q000633:toxicity*; D015034:Zinc Oxide / Q000737:chemistry / Q000633:toxicity*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; C071525:FtsZ protein, Bacteria; D011135:Polysaccharides, Bacterial; D012989:Soil Pollutants; D049452:Green Fluorescent Proteins; D015034:Zinc Oxide