Comparative Eco-Toxicities of Nano-ZnO Particles under Aquatic and Aerosol Exposure Modes BING WU, YIN WANG, YI-HSUAN LEE, ANGELA HORST, ZHIPENG WANG, DA-REN CHEN, RADHAKRISHNA SURESHKUMAR, † AND YINJIE J. TANG* Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, Missouri 63130 Received October 6, 2009. Revised manuscript received January 6, 2010. Accepted January 12, 2010. The antimicrobial activity of ZnO nanoparticles (NPs) was investigated under aquatic and aerosol exposure modes. ZnO NPs in aquatic media aggregated to micrometer-sized particles and did not interact with microorganisms effectively. Hence, the inhibition of microbial growth by nano-ZnO NPs (e.g., Mycobacterium smegmatis and Cyanothece 51142) in aquatic media was mainly attributable to dissolved zinc species. Shewanella oneidensis MR-1 and Escherichia coli were able to produce large amounts of extracellular polymeric substances, and their growth was not inhibited by ZnO NPs in aquatic media, even at high concentrations ( >40 mg/L). On the other hand, when ZnO NPs were electrosprayed onto an E. coli biofilm so that NPs could be directly deposited onto the cell surface, the aerosol exposure dramatically reduced cellular viability. For example, an electrospray of ZnO NPs (20 nm) reduced the total number of viable E.coli cells by 57% compared to the control case, in which we electrosprayed only the buffer solution. However, electrospraying large-sized ZnO particles (480 nm) or nonsoluble TiO 2 NPs (20 nm) caused much less lethality to E. coli cells. The above observation implies that the aerosol method of exposing ZnO NPs to biological systems appears to have a much higher antimicrobial activity, and thus may lead to practical applications of employing a novel antimicrobial agent for airborne disease control. Introduction ZnO nanoparticles (NPs) are widely used as pigments, semiconductors, sunscreens, and food additives. To deter- mine the potential eco-toxicity of ZnO NPs, researchers have investigated their toxicological properties, fate, and transport in the environment (1–3). Injurious effects of ZnO NPs upon a variety of organisms in aquatic environments have recently been reported (1, 4–6). Several studies indicate that the dissolved Zn 2+ from ZnO NPs in the aquatic environment causes these eco-toxicities. Other studies have shown that metal NPs may be more toxic than either their ionic forms or their parent compounds (7, 8). NPs tend to aggregate in aquatic environments to form micrometer-sized particles, and this state of dispersion reduces the influences of particle size, particle shape, and surface charge on the NPs’ eco- toxicity (9, 10). Therefore, a variety of methods have been used for dispersing NPs, such as the addition of solvents (or dispersants), the use of ultrasonication, and the act of shaking (or stirring). These methods, however, may undermine the validity of NPs toxicity studies (8). For example, ultrasoni- cation can change NP’s structures, and solvents may themselves be toxic to the tested microorganisms. Comparatively, less effort has been focused on microbial responses to NPs under aerosol exposure modes. Such exposure modes enable direct interactions between cells and NPs, and thus nanoassociated toxicity can be more easily observed with this method than with the aquatic exposure mode. Rothen-Rutishauser and his coresearchers have employed nanoparticle fabrication systems to study the direct exposure of CeO 2 NPs to lung cell cultures in order to mimic the toxic effect of airborne NPs (11). Their study clearly reveals a dose-dependent cellular response to the airborne engi- neered nanoparticles. In this study, we are interested in the aerosol exposure of microorganisms to NPs and in exploring whether such exposure modes can enhance NPs’ antimi- crobial activity. We applied a novel electrospray technique (formally called “electro-hydrodynamic atomization”) to ensure the ZnO NPs were suspended in airborne droplets (12–15). The spray technique can produce droplets with varying diameters in the nanometer scale (16). By controlling the electrical charge of the droplets produced in the elec- trospray, ZnO NPs can be dispersed in nanoscale with minimal aggregation. This approach mimics the exposure of microorganisms to airborne NPs and allows the direct deposit of ZnO NPs onto bacterial surfaces. The antimicrobial activity of ZnO NPs in the aerosol exposure mode suggests that they potentially can control airborne disease (e.g., influenza, tuberculosis, and pneumonia), prevent bacterial fouling, and reduce the risk of NP bioaerosol attack (17). Materials and Methods Cultivation of Organisms with NPs. Five microbial species were used: one pathogenic bacterium (nonvirulent species Mycobacterium smegmatis), one metal reducing bacterium (Shewanella oneidensis MR-1), one blue-green algae (Cyan- othece 51142), one yeast (Saccharomyces cerevisiae), and one enterobacterium (Escherichia coli strain BL21). M. smegmatis was grown in a Sauton liquid medium at 37 °C(18); S. oneidensis MR-1 was grown in a MR-1 medium at 30 °C(19); E. coli was grown in a M9 medium at 37 °C; Cyanothece 51142 was grown in a ASP2 medium at 30 °C under continuous light (50 µmol photons m -2 s -1 )(20); and S. cerevisiae was grown in a yeast synthetic-defined (SD) medium at 30 °C. All cultures (50 mL) were grown in flasks shaken at 150 rpm in the dark, except for Cyanothece 51142. ZnO (20 nm and 480 nm), and TiO 2 (20 nm) nanoparticles were obtained from NanoAmor (Houston, TX), and were dispersed in sterilized DI water to make 1 g/L solution. The initial cell concentrations in the cultures had an optical density (OD) ∼0.1 (i.e., the early log phase). Then, different amounts of ZnO NP stock solution were added into the cell cultures, and cell growth was monitored using a spectrometer (Genesys, Thermo Scientific, U.S.) at a wavelength of 600 nm (for M. smegmatis, S. oneidensis MR-1, S. cerevisiae, and E. coli) and 730 nm (for Cyanothece 51142). The tested NPs also absorbed light at OD 600 and OD 730 ; thus the actual cell density was obtained based on * Corresponding author phone: 314-935-3441; e-mail: yinjie. tang@seas.wustl.edu. † Present Address: Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, NY 13244. Environ. Sci. Technol. 2010, 44, 1484–1489 1484 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 4, 2010 10.1021/es9030497  2010 American Chemical Society Published on Web 01/26/2010