Effects of engineered cerium oxide nanoparticles on bacterial growth and viability

Abstract

Interest in engineered nanostructures has risen in recent years due to their use in energy conservation strategies and biomedicine. To ensure prudent development and use of nanomaterials, the fate and effects of such engineered structures on the environment should be understood. Interactions of nanomaterials with environmental microorganisms are inevitable, but the general consequences of such interactions remain unclear, due to a lack of standard methods for assessing such interactions. Therefore, we have initiated a multianalytical approach to understand the interactions of synthesized nanoparticles with bacterial systems. These efforts are focused initially on cerium oxide nanoparticles and model bacteria in order to evaluate characterization procedures and the possible fate of such materials in the environment. The growth and viability of the Gram-negative species Escherichia coli and Shewanella oneidensis, a metal-reducing bacterium, and the Gram-positive species Bacillus subtilis were examined relative to cerium oxide particle size, growth media, pH, and dosage. A hydrothermal synthesis approach was used to prepare cerium oxide nanoparticles of defined sizes in order to eliminate complications originating from the use of organic solvents and surfactants. Bactericidal effects were determined from MIC and CFU measurements, disk diffusion tests, and live/dead assays. For E. coli and B. subtilis, clear strain- and size-dependent inhibition was observed, whereas S. oneidensis appeared to be unaffected by the particles. Transmission electron microscopy along with microarray-based transcriptional profiling was used to understand the response mechanism of the bacteria. Use of multiple analytical approaches adds confidence to toxicity assessments, while the use of different bacterial systems highlights the potential wide-ranging effects of nanomaterial interactions in the environment.

FIG. 1.

TEM and AFM analyses of the four different sizes of cerium oxide nanoparticles. (a to d) TEM images of the smallest to the largest CeO₂ particles, referred to as samples A (a), B (b), C (c), and D (d). (e to h) AFM images of the particles; (e) sample A and its topographic image that is used to accurately measure height; (f to h) deflection images of the sample B, C, and D particles, respectively. The deflection images better represent changes in surface morphology of the CeO₂ nanoparticles.

FIG. 2.

Zeta potential and dynamic light scattering measurements of the B sample of cerium oxide nanoparticles. The zeta potential (a) and hydrodynamic sizes (b) of the B sample of cerium oxide nanoparticles in water and M9, B. subtilis minimal, and HBA media under different pH conditions are shown. Similar results were obtained with the other nanoparticle samples.

FIG. 3.

Diameter of zone of inhibition and MIC assays for E. coli and B. subtilis induced by different-sized sample A, B, C, and D cerium oxide nanoparticles. (a) Measurement of the diameter of the zone of inhibition (in millimeters) was carried out by disk diffusion assay, and the results are shown in the form of bar graphs for E. coli (left panel) and B. subtilis (right panel). (b to e) Dynamic growth curves for E. coli (left panels) and B. subtilis (right panels) in their respective minimal media treated with different sized cerium oxide nanoparticles at various concentrations (▪, cells alone; •, 50 mg/liter; ▴, 100 mg/liter; ▾, 150 mg/liter). (b) sample a; (c) sample B; (d) sample C; (e) sample D.

FIG. 4.

Representative TEM images showing the interaction of E. coli and the B sample of cerium oxide nanoparticles at different magnifications. The images shows the results of incubating nanoparticles with logarithmic-phase growing bacteria for 30 min at 37°C with shaking, followed by placing a droplet on the EM grid for 7 min, rinsing in water to remove unbound bacteria and particles, and imaging. Particles apparently stick to the bacterial surfaces but are not internalized by E. coli.

FIG. 5.

Proportional Venn diagram analysis of significant differentially expressed genes. Nano-Control, sample B CeO₂ nanoparticle-Milli-Q control differences; Nano-Salt, sample B CeO₂ nanoparticle-CeCl₃ control differences; Conrol-Salt, Milli-Q control-CeCl₃ control differences. For a complete list of genes within categories A to G, see Table S1 in the supplemental material.