Grantee Research Project Results
Final Report: Microbial Impacts of Engineered Nanoparticles
EPA Grant Number: R832534Title: Microbial Impacts of Engineered Nanoparticles
Investigators: Alvarez, Pedro J. , Wiesner, Mark R.
Institution: Rice University , Duke University
EPA Project Officer: Hahn, Intaek
Project Period: December 15, 2005 through December 14, 2008
Project Amount: $375,000
RFA: Exploratory Research: Nanotechnology Research Grants Investigating Environmental and Human Health Effects of Manufactured Nanomaterials: A Joint Research Solicitation - EPA, NSF, NIOSH (2005) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals
Objective:
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.
Summary/Accomplishments (Outputs/Outcomes):
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.
Comparative photoactivity and microbial toxicity of fullerenes and titanium dioxide
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.
Journal Articles on this Report : 12 Displayed | Download in RIS Format
Other project views: | All 16 publications | 15 publications in selected types | All 13 journal articles |
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Badireddy AR, Hotze EM, Chellam S, Alvarez P, Wiesner MR. Inactivation of bacteriophages via photosensitization of fullerol nanoparticles. Environmental Science & Technology 2007;41(18):6627-6632. |
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Brunet L, Lyon DY, Zodrow K, Rouch J-C, Caussat B, Serp P, Remigy J-C, Wiesner MR, Alvarez PJJ. Properties of membranes containing semi-dispersed carbon nanotubes. Environmental Engineering Science 2008;25(4):565-576. |
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Fang J, Lyon DY, Wiesner MR, Dong J, Alvarez PJJ. Effect of a fullerene water suspension on bacterial phospholipids and membrane phase behavior. Environmental Science & Technology 2007;41(7):2636-2642. |
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Hotze EM, Labille J, Alvarez P, Wiesner MR. Mechanisms of photochemistry and reactive oxygen production by fullerene suspensions in water. Environmental Science & Technology 2008;42(11):4175-4180. |
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Klaine SJ, Alvarez PJJ, Batley GE, Fernandes TF, Handy RD, Lyon DY, Mahendra S, McLaughlin MJ, Lead JR. Nanomaterials in the environment: behavior, fate, bioavailability, and effects. Environmental Toxicology and Chemistry 2008;27(9):1825-1851. |
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Li Q, Mahendra S, Lyon DY, Brunet L, Liga MV, Li D, Alvarez PJJ. Antimicrobial nanomaterials for water disinfection and microbial control: potential applications and implications. Water Research 2008;42(18):4591-4602. |
R832534 (Final) |
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Lyon DY, Adams LK, Falkner JC, Alvarez PJJ. Antibacterial activity of fullerene water suspensions: effects of preparation method and particle size. Environmental Science & Technology 2006;40(14):4360-4366. |
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Lyon DY, Brunet L, Hinkal GW, Wiesner MR, Alvarez PJJ. Antibacterial activity of fullerene water suspensions (nC60) is not due to ROS-mediated damage. Nano Letters 2008;8(5):1539-1543. |
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Lyon DY, Alvarez PJJ. Fullerene water suspension (nC60) exerts antibacterial effects via ROS-independent protein oxidation. Environmental Science & Technology 2008;42(21):8127-8132. |
R832534 (Final) |
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Lyon DY, Brown DA, Alvarez PJJ. Implications and potential applications of bactericidal fullerene water suspensions: effect of nC60 concentration, exposure conditions and shelf life. Water Science and Technology 2008;57(10):1533-1538. |
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Wiesner MR, Lowry GV, Alvarez P, Dionysiou D, Biswas P. Assessing the risks of manufactured nanomaterials. Environmental Science & Technology 2006;40(14):4336-4345. |
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Zodrow K, Brunet L, Mahendra S, Li D, Zhang A, Li Q, Alvarez PJJ. Polysulfone ultrafiltration membranes impregnated with silver nanoparticles show improved biofouling resistance and virus removal. Water Research 2009;43(3):715-723. |
R832534 (Final) |
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Supplemental Keywords:
Health, Scientific Discipline, Health Risk Assessment, Environmental Chemistry, Risk Assessments, Ecological Risk Assessment, anthropogenic stress, ecotoxicogenomics, bioavailability, nanotechnology, nanomaterials, microbial riskProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.