Grantee Research Project Results

Responsible usage of nanomaterials in commercial products and environmental applications, and prudent management of the associated risks, require an understanding of nanoparticle mobility, bioavailability and ecotoxicology. This project seeks to elucidate processes governing the transport and microbial impacts of two classes of catalytic nanomaterials in soil-water systems: fullerenes and metallic nanoparticles (e.g., TiO₂). Specific tasks include to

 

(1)   characterize nanomaterials size, shape, functionality, reactivity, aggregation, deposition potential, and bioavailability;

(2)   screen nanomaterials of varying sizes and properties for bactericidal activity;

(3)   discern bacterial physiologic characteristics that confer resistance (or susceptibility) to catalytic nanomaterials;

(4)   evaluate the potential for fullerene biotransformation by reference bacteria and fungi; and

(5)   assess the impact of simulated nanomaterial releases on microbial diversity and community structure.

 

The relevance of this work to the EPA mission is related to the fact that microorganisms are the foundation of all ecosystems and are often the basis for food chains and the main agents of biogeochemical cycles. Thus, understanding their interactions with engineered nanomaterials is important to ensure that nanotechnology improves material and social conditions without exceeding the ecological capabilities that support them. At the conclusion of this project, we will have an improved understanding of the chemical and physical factors that control nanoparticle mobility and bioavailability, and their impacts on microbial activities, diversity, and community structure. This will benefit risk assessment and management efforts, and may contribute indirectly to the development of nanotechnology-based disinfection and biofouling control strategies.

ROS production by C₆₀ and functionalized C₆₀.  The fullerenes were evaluated for photochemical ROS production and photo-induced antibacterial activity towards E. coli.  Kinetics of ROS production by nC₆₀ preparations were compared with those of TiO₂, a well-characterized photocatalyst. Dispersion of C₆₀ in water was accomplished by the following methods: (1) THF solvent exchange (nC₆₀/THF) and long-term stirring (nC₆₀/Aq), (2) encapsulation of C₆₀ with PVP, and (3) a multihydroxylation of C₆₀ (fullerol).  To identify ROS produced during the photoexcitation of the fullerenes in water, singlet oxygen sensor green (SOSG) reagent, 2,3- bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) dye, and p-chlorobenzoic acid (pCBA) were used as indicators for ¹O₂, O₂^•-, and OH^•, respectively. As expected, TiO₂ produced all types of ROS in the UV-illuminated aqueous suspensions, though generated only O₂^•- in bacterial growth mediumOne the other hand, nC₆₀/THF and nC₆₀/Aq did not present any significant photoreactivity in both water and bacterial growth medium. C₆₀/PVP and fullerol (which were far less aggregated than nC₆₀/THF and nC₆₀/Aq) generated ¹O₂ and O₂^•- in the growth medium, while fullerol yielded ¹O₂ to in ultra-pure water. The ROS production by C₆₀ fullerenes was not correlated with their antibacterial activity, which suggests that ROS may not be the primary agent of toxicity of fullerenes-water suspensions.

Toxicity Mechanism of nC₆₀.  We tested the hypothesis that nC₆₀ behaves as an oxidant and exerts ROS-independent oxidative stress on bacterial cells.  Measurements of oxidation reduction potentials (ORP) of different nC₆₀ suspensions indicated that nC₆₀ acted as an oxidant indeed.  A lipid peroxidation assay was previously described to assess ROS damage to cell walls.  In this assay, thiobarbituric acid (TBA) reacted with malondialdehyde (MDA), an oxidation product of polyunsaturated fatty acids to produce a color change indicating lipid peroxidation.  However, TBA has also been known to yield a color after reacting with other chemicals, and, in our studies, nC₆₀ reacted directly with TBA to produce color, with or without the presence of cells, indicating that this assay was confounded by nC₆₀ itself.  In the light of our data invalidating ROS-mediated toxicity of nC₆₀, other mechanisms were explored using a variety of assays to observe the effects of nC₆₀ on cells.  A propidium iodide assay was used in conjunction with flow cytometry to detect any damage to the cell membrane; however, no damage to the cell membrane was observed in either the Gram-positive Bacillus subtilis or the Gram-negative Escherichia coli.  Using the BacLight Membrane Potential kit to detect any changes in membrane potential, essential to respiration, only B. subtilis showed a change in membrane potential after exposure to nC₆₀, although both B. subtilis and E. coli were similarly affected.  Bacterial reductase activity was inhibited upon nC₆₀ exposure, which indicated that the electron transport chain was affected under aerobic conditions.  Additionally, results from experiments conducted with a thiol oxidation kit indicate that nC₆₀ can oxidize proteins, but only affects proteins on the outside of the cell, where they interact with nC₆₀ directly.  Thus, we concluded that there nC₆₀ results in oxidative damage that is not mediated by ROS but most likely is a result of oxidative stress on direct contact of nC₆₀ with the cells.

Nanosize anatase (TiO₂) and fullerenes are two nanomaterials whose photoactivity has been scrutinized for its possible involvement in cytotoxicity as well as its potential applications. We compared the singlet oxygen (¹O₂), superoxide (O₂^-), and hydroxyl radical (OH^•) generation capacity of different fullerene water suspensions to that of a nano-TiO₂ (Degussa P25) in both ultra-pure water and a microbial growth medium. In ultra-pure water, nano-TiO₂ produced primarily OH^•, and fullerol produced only ¹O₂. In the mineral medium, nano-TiO₂’s OH^• were quenched, but O₂^- production was enhanced. More ¹O₂ was generated by PVP/C₆₀ and fullerol than by nano-TiO₂, whereas O₂^- was generated by nano-TiO₂, PVP/C₆₀ and fullerol, in order of decreasing activity. However, in none of the media, the C₆₀ aggregates obtained by extensive stirring (Aq/nC₆₀) or exchange of solvent (THF/nC₆₀) were able to produce reactive oxygen species (ROS). Additional tests showed that nano-TiO₂ exhibited antibacterial activity in presence of light but not in the dark. Other nanoparticles were either not antibacterial at all (fullerol, Aq/nC₆₀) or had similar antibacterial activity (THF/nC₆₀, and PVP/C₆₀) regardless of light. These results demonstrate the lack of correlation between ROS production and toxicity for the fullerenes, and suggest that O₂^- contributed to nano-TiO₂ phototoxicity. Based on ROS speciation in the two media tested and the oxidation potential of each ROS, we recommend that fullerol and encapsulated fullerenes be applied in biomedical applications and to accompany water treatment targeting more specifically pollutants or microorganisms more sensitive to either superoxide or singlet oxygen, whereas nano-TiO₂ would be more efficient for water treatment in general, involving UV or solar energy, to enhance disinfection or contaminant oxidation.