The aim of this work was to evaluate the photokilling efficiency of synthesized titanium dioxide nanoparticles in suspension. Two strains of Escherichia coli, Lactobacillus casei rhamnosus and Staphylococcus aureus were used as probes to test the photokilling activities of the nanoparticles. The toxicity effects of TiO<SUB> 2</SUB> nanoparticles on the environment were determined by a standard test using gram-negative bioluminescent bacteria Vibrio fischeri. The antimicrobial activity of these nanoparticles (NPs) was then investigated versus NPs concentration, UV irradiation time and micro- organism strains. We evaluated the LC50 values of the nanoparticles suspension by counting the Colony-Forming Units. Results highlighted the differences in bacteria sensitivity facing photokilling treatment induced by the irradiation of anatase TiO<SUB> 2</SUB> nanoparticles suspension. At the concentration of 1 g·L <sup>-1</sup> TiO<SUB> 2</SUB>, tested bacteria were killed after 30 minutes of photo-treatment. Using different TiO<SUB> 2</SUB> concentrations, the Staphylococcus aureus gram-positive/catalase-positive bacteria were more resistant than gram-negative/catalase-positive ones or gram-positive/catalase-negative bacteria. An effect of UV irradiation was evaluated by the quantification of hydrogen peroxide generated by the photolysis of water molecules in presence of the nanoparticles with or without the most resistant bacterium (S. aureus). After 30 minutes with UV irradiation in these two conditions, the concentration of hydrogen peroxide was 35 μM in presence of 1.2 g·L<sup> -1</sup> TiO<SUB> 2</SUB> suspension. This result suggested that the resistance mechanism of S. aureus was not due to an extracelullar H<SUB> 2</SUB>O <SUB>2</SUB> enzymatic degradation.
Photokilling of pathogen species is a promising alternative compared to conventional disinfection process. In particular, when chemical cleaning products are not effective or dangerous, a disinfection protocol based on the irradiation of photoactive species can be interesting. Contrary to other cleaning treatments, such as chlorination [
In this study, we synthesized an original and stable anatase-crystallized suspension of TiO2 nanoparticles. Escherichia coli strains LE392 and ETEC H10407 (gram-negative/catalase-positive bacteria), Lactobacillus casei rhamnosus strain Lcr35® (gram-positive/catalase-negative bacteria) and Staphylococcus aureus (SA51, gram-posi- tive/catalase-positive bacteria) were used as probes to test the photokilling efficiency of the nanoparticles in suspension. In particular, the resistance behaviour of different bacteria strains was evaluated using LC50 tests, focusing on two different parameters: the bacteria wall thickness (gram+ or gram−) and the presence or absence of the catalase gene (catalase+ or catalase−). Bioluminescent tests were performed to investigate the environmental toxicity of TiO2 in suspension. The quantification of H2O2 allowed a better understanding of the inactivation mechanism involved in the photokilling process.
The precursor solution was 10 mL titanium IV isopropoxide supplied by Sigma Aldrich mixed with 10 mL of anhydrous isopropanol (from Sigma) using a magnetic stirrer at 300 rpm. The titanium alkoxide reactivity was lowered by the use of acetylaceton. The spontaneous hydrolysis of the titanium isopropoxide was obtained by the quick addition of 75 mL of acidified water. The reacting medium was then heated to 100˚C under reflux for almost 8 hours (peptidization process). After this step, the dispersion was cooled down to room temperature, approximately 20˚C. A clear, yellow, anatase crystallized nanoparticles suspension was obtained, and stored in the dark.
The morphology and the particle sizes were characterized using a Philips CM 20 transmission electron microscope (TEM). The accelerating voltage was 200 kV. The samples were dispersed in methanol by ultrasonication. A drop of the suspension was then laid on a carbon-coated grid and dried. Selected Area Electron Diffraction (SAED) was performed to determine the crystalinity of the structure. The interplanar spacings were evaluated from the SAED patterns using the following formula:
λL = Rd (1)
where λL is the constant of the microscope, R is the ring radius, and d is the interplanar spacing. The constant of the microscope was calculated by measuring the radius of a gold standard pattern whose interplanar spacings were well documented in scientific publications [
Four micro-organisms were used for photokilling experiments: Escherichia coli LE392, Enterotoxigenic Escherichia coli H10407, Lactobacillus casei rhamnosus Lcr35® and Staphylococcus aureus (SA51). These bacteria have a size comprised between 0.5 and 5 µm. E. coli cells were cultured at 37˚C for 24 h in Nutrient Broth medium at pH 7.2 (Biokar diagnostics) containing Tryptone (10 g・L−1), Meat extract (5 g・L−1) and Sodium Chloride (5 g・L−1) after 12 h of pre-culture in the same conditions. Lactobacillus casei rhamnosus Lcr35® was cultured in De Man, Rogosa, Sharpe (MRS) medium (Bio-Rad, Mitry Mory, France) and S. aureus in Brain Heart Broth (Brain Heart Infusion 17.5 g・L−1, Pancreatic digest of gelatin 10 g・L−1, Sodium Chloride 5 g・L−1, Disodium phosphate 2.5 g・L−1, Glucose 2 g・L−1, Biokar diagnostics) under the same conditions than E. coli strains. Cells were centrifuged at 2500 g for 15 min at 4˚C and the pellet was re-suspended in de-ionized water to prevent unintentional increase in cell numbers. The initial population of bacteria was determined by enumeration with a Petroff-Hausser Counting Chamber.
The Microtox® Procedure employs the bioluminescent marine gram-negative bacterium Vibrio fischeri as test organism. The bacteria are exposed to a range of concentration of the TiO2 in suspension being tested. The reduction in intensity of light emitted from the bacteria is measured along with standard solutions and control samples. Toxicity is, then, inversely proportional to the intensity of the light emitted after contact with the toxic substances. The change in light output and concentration of the toxicant produce a dose/response relationship. The results are normalized and the EC50 (concentration producing a 50% reduction in light) is calculated.
The basic test protocol (consisting of four test dilutions) was carried out to evaluate the ecotoxicity of the medium containing TiO2 nanoparticles. All tests were performed using the Microtox 500 Analyser, and bioluminescence measurements were monitored at 0, 5 and 15 min of exposure. The effective concentrations causing 50% of bioluminescence inhibition were computed using the software for Microtox Omni Azur (AZUR environmental, 1998). Toxicity tests were performed in triplicate each week during a two months period and the results are expressed in mg・L−1.
For inactivation kinetics measurements, an amount of 20 mL of de-ionized water was inoculated with Escherichia coli LE392 or Enterotoxigenic Escherichia coli H10407 suspension in order to achieve a concentration of 106 CFU・mL−1 (Colony-Forming Unit by mL). This suspension was placed in a Petri plate with TiO2 nanoparticles to achieve a final concentration in TiO2 of 1 g・L−1. The slurries were continuously mixed and irradiated with UV (polychromatic fluorescent UV lamps (©Philips TLD 8 W) providing a total power of 48 W, in a configuration delivering 1.5 mW・cm−2 at the liquid surface). A complete mixing was done with a sterilized Teflon magnetic stir bar placed in the Petri dish with a speed of 200 rpm.
Sampling of the solutions was done at requisite time intervals (from 0 to 30 min) by pipetting 1 mL from the suspension and serially diluted in 9 mL of Ringer’s solution. After sufficient mixing, 100 µL aliquots of each dilution were plated onto solid Nutrient Gelose medium (Biokar diagnostics) with agar 15 g・L−1. Colony-Forming Units were counted after overnight incubation at 37˚C. All experiments were made in aseptic conditions to prevent any contamination in the media. The counts from three independent experiments corresponding to a particular sample were averaged. The method used for the LC50 tests was similar to that used for inactivation kinetics and was performed on all bacteria strains with nanoparticles concentrations from 50 to 1200 mg・L−1 TiO2 under 30 min UV irradiation at 1.5 mW・cm−2.
Generation of hydrogen peroxide by TiO2 nanoparticules in an aqueous liquid suspension was determined as described by Batdorj et al. [
Stable titanium dioxide nanoparticles in suspension are fabricated using a derivate sol gel process.
nanoparticles in an aqueous liquid. Taking into account that the pH of liquid carrier is low, the TiO2 mineral oxide nanoparticles have a positive surface charge.
A TEM picture and the associated SAED pattern of our as-synthesized sample are presented in
The SAED patterns of the most intense spots are shown in
The Microtox® test has been routinely applied to treated waste waters or single compounds and mixtures of inorganic and organic compounds [
We have demonstrated the toxicity of our nanoparticle suspension in the dark on a very sensitive bacterium, Vibrio fischeri.
As in previous studies on Escherichia coli LE392 [
Interplanar distance from the SAED pattern (Å) | 3.57 | 2.41 | 1.93 | 1.70 |
---|---|---|---|---|
Theoretical distance for the anatase phase (Å) | 3.51 | 2.33 | 1.89 | 1.66 |
Corresponding Miller indice | (101) | (103) | (200) | (211) |
after only 1 hour of treatment with 1 g・L−1 TiO2 suspension under UV irradiation, we could wonder what happens during this time duration.
Freshly grown bacterial cultures (106 CFU・mL−1) were treated with 1 g・L−1 of TiO2 and irradiated with UV (1.5 mW・cm−2). This experiment was carried out in triplicate. Wang et al. [
The 30 minutes-LC50 tests were then performed on all strains in order to make a comparison between bacteria differing in cell wall structure and detoxification system implicating the catalase enzyme (
Concentration-dependent mortality in E. coli exposed to TiO2 suspension under 30 minutes UV irradiation (1.5 mW・cm−2) showed a linear profile for both strains at a concentration ranging from 100 to 600 mg・L−1 (
The fact that TiO2 nanoparticles showed a lower effect on S. aureus than on the other ones, under the same conditions, indicates that the resistance of bacteria to TiO2 nanoparticles is species-dependent. These differences might be due to different structural properties of cell wall and/or a higher self-defense property [
Bacteria | Gram | Catalase | LC50 (mg・L−1) |
---|---|---|---|
E. coli LE392 | negative | positive | 340 |
Enterotoxigenic E. coli | negative | positive | 281 |
L. casei rhamnosus | positive | negative | 195 |
S. aureus | positive | positive | 585 |
results [
Major constituents of the cell wall are each specific strains and the surface charge of the bacteria is associated with the presence of the ionized groups of the macromolecules [
There are reports in the literature that show that electrostatic attraction between negatively charged bacterial cells and positively charged nanoparticles is crucial for the activity of nanoparticles as bactericidal materials. Nanoparticles are capable of penetrating bacterial cells and act as a catalyst, to inactivate enzymes that micro- organisms need for their metabolism by interacting with thiol groups of proteins, disrupt bacterial membranes and also affect DNA replication [
In our study, we have to take into account the presence or absence in cells of an enzyme responsible for catalyzing the breakdown of hydrogen peroxide into water and molecular oxygen: catalase [
TiO2 is a semiconductor [
With the aim of evaluating the H2O2 production capacity by TiO2 nanoparticles in the dark or under UV irradiation after 30 minutes, we measured concentration of this molecule with regard with different nanoparticles concentrations (0 from 1200 mg・L−1,
For this experiment, we chose S. aureus because it was the most resistant bacterium among the four tested. Its resistance may be due to a detoxification capacity of the external environment by a catalase activity. Effectively, in order to counteract excess ROS, various antioxidant mechanisms are activated in the organisms. The initial mechanisms that act to adjust antioxidant levels to protect the cells include changes in antioxidant gene expression [
We observed (
Under UV irradiation, H2O2 concentration obtained was significantly greater than in dark condition. The maximum concentration (35 ± 1.66 µM hydrogen peroxide) was achieved with 1200 mg・L−1 of nanoparticles without S. aureus. As in the dark condition, the bacteria did not change the content of H2O2 in their extracellular environment.
The greatest resistance of S. aureus to TiO2 nanoparticles under UV irradiation is probably due to an intracellular detoxification process and wall thickness properties.
In this study, we synthesized stable anatase titanium dioxide nanoparticles in suspension. We evaluated the environmental toxicity of suspension using Microtox® test. The Microtox® test using Vibrio fischeri has classified our nanoparticles as harmful to aquatic micro-organisms. The hydrogen peroxide quantification indicated that H2O2 was involved in the biological mechanism. The comparison between the bacteria strains showed a higher resistance with S. aureus than with E. coli and Lcr35®. This resistance may be due to the presence of the catalase gene in its genome and its thicker wall.
However, further studies are needed in order to elucidate mechanisms of toxicity induced by our TiO2 nanoparticles, so it could be interesting to determine intracellular ROS concentration, lipid peroxidation level, membrane integrity and DNA damage. Gene expression analysis by RT-qPCR and/or RNA-Seq will also permit us to assess all the effects of our nanoparticles on the different metabolic pathways and especially on the oxidative pathway.
The authors acknowledge the University of Auvergne for its financial support and the company Probionov for the gift of Lactobacillus casei rhamnosus Lcr35® and Enterotoxigenic E. coli H10407. The authors acknowledge Yves Sibaud and Michelle Conry for their technical support.
MurielBonnet,ChristopheMassard,PhilippeVeisseire,OlivierCamares,Komla OscarAwitor, (2015) Environmental Toxicity and Antimicrobial Efficiency of Titanium Dioxide Nanoparticles in Suspension. Journal of Biomaterials and Nanobiotechnology,06,213-224. doi: 10.4236/jbnb.2015.63020