Differential growth of and nanoscale TiO2 accumulation in Tetrahymena thermophila by direct feeding versus trophic transfer from Pseudomonas aeruginosa.

Nanoscale titanium dioxide (TiO2) is increasingly used in consumer goods and is entering waste streams, thereby exposing and potentially affecting environmental microbes. Protozoans could either take up TiO2 directly from water and sediments or acquire TiO2 during bactivory (ingestion of bacteria) of TiO2-encrusted bacteria. Here, the route of exposure of the ciliated protozoan Tetrahymena thermophila to TiO2 was varied and the growth of, and uptake and accumulation of TiO2 by, T. thermophila were measured. While TiO2 did not affect T. thermophila swimming or cellular morphology, direct TiO2 exposure in rich growth medium resulted in a lower population yield. When TiO2 exposure was by bactivory of Pseudomonas aeruginosa, the T. thermophila population yield and growth rate were lower than those that occurred during the bactivory of non-TiO2-encrusted bacteria. Regardless of the feeding mode, T. thermophila cells internalized TiO2 into their food vacuoles. Biomagnification of TiO2 was not observed; this was attributed to the observation that TiO2 appeared to be unable to cross the food vacuole membrane and enter the cytoplasm. Nevertheless, our findings imply that TiO2 could be transferred into higher trophic levels within food webs and that the food web could be affected by the decreased growth rate and yield of organisms near the base of the web.

An assessment of fluorescence- and absorbance-based assays to study metal-oxide nanoparticle ROS production and effects on bacterial membranes.

The production and inevitable release of engineered nanoparticles requires rapid approaches to screen for their potential effects in environmental organisms, including bacteria. In bacteria, engineered nanoparticle effects can initiate at the cell membrane, for example by structurally damaging membranes or inhibiting energy transduction. Commercially available fluorescence- and absorbance-based assays could allow for rapidly assaying engineered nanoparticle effects on bacterial membranes, but there are limitations, including that: 1) assays are not currently configured to operate as part of a comprehensive high-throughput screening system, since assay conditions vary widely and formats are mostly high-volume and thus low-throughput, and; 2) engineered nanoparticles can interfere with assay reagents or function, yielding false-negative or -positive outcomes. Here, key assays to study reactive oxygen species (total ROS, and superoxide) production, and impacts on bacterial membrane integrity, membrane potential, and electron transport chain activity, are assessed for their potential use as a comprehensive system to test for nanoparticle effects in bacteria. To address (1), assays are adapted for simultaneous use in 96-well microplates under harmonized conditions. To address (2), a general scheme to test for engineered nanoparticle interferences with assay reagents and function is conceived, and used to study assay interferences by three nanoscale metal-oxides: nano-TiO2 , nano-CeO2 , and nano-ZnO. The results show that the selected assays can be used as a suite, and that nanoparticle interferences, when they occur, can be systematically investigated and often accounted for.

Soybean susceptibility to manufactured nanomaterials with evidence for food quality and soil fertility interruption.

Based on previously published hydroponic plant, planktonic bacterial, and soil microbial community research, manufactured nanomaterial (MNM) environmental buildup could profoundly alter soil-based food crop quality and yield. However, thus far, no single study has at once examined the full implications, as no studies have involved growing plants to full maturity in MNM-contaminated field soil. We have done so for soybean, a major global commodity crop, using farm soil amended with two high-production metal oxide MNMs (nano-CeO(2) and -ZnO). The results provide a clear, but unfortunate, view of what could arise over the long term: (i) for nano-ZnO, component metal was taken up and distributed throughout edible plant tissues; (ii) for nano-CeO(2), plant growth and yield diminished, but also (iii) nitrogen fixation--a major ecosystem service of leguminous crops--was shut down at high nano-CeO(2) concentration. Juxtaposed against widespread land application of wastewater treatment biosolids to food crops, these findings forewarn of agriculturally associated human and environmental risks from the accelerating use of MNMs.

Nanomaterials in the environment: from materials to high-throughput screening to organisms.

One of the challenges in the field of nanotechnology is environmental health and safety (EHS), including consideration of the properties of engineered nanomaterials (ENMs) that could pose dangers to the environment. Progress in the field of nanomaterial development and nanotoxicology was presented at the International Conference on the Environmental Implications of Nanotechnology at the California NanoSystems Institute (CNSI) on the UCLA campus on May 11-14, 2010. This event was cohosted by the University of California Center for the Environmental Implications of Nanotechnology (UC CEIN) and the Center for the Environmental Implications of NanoTechnology (CEINT) based at Duke University. Participants included scientists and scholars from various backgrounds, including chemistry, biology, engineering, nanomaterial science, toxicology, ecology, mathematics, sociology, and policy makers. The topics of discussion included safety evaluation of ENMs from an environmental perspective, nanotoxicology, ecotoxicology, safe design of ENMs, environmental risk assessment, public perception of nanotechnology, application of ENMs in consumer products, and many more. The UC CEIN presented data on their predictive toxicological approach to the assessment of ENM libraries, which were designed and synthesized to develop an understanding of the material properties that could lead to hazard generation at the cellular and organismal levels in the environment. This article will focus on the first metal oxide ENM library that was introduced to harmonize research activities in the UC CEIN, with particular emphasis on the safety assessment of ZnO on cells and organisms. Methods of decreasing the observed toxic effects will also be discussed as an integral component of the UC CEIN's activity in developing safer nanomaterials to lessen their environmental impacts.

Dispersion of TiO2 nanoparticle agglomerates by Pseudomonas aeruginosa.

Engineered nanoparticles are increasingly incorporated into consumer products and are emerging as potential environmental contaminants. Upon environmental release, nanoparticles could inhibit bacterial processes, as evidenced by laboratory studies. Less is known regarding bacterial alteration of nanoparticles, including whether bacteria affect physical agglomeration states controlling nanoparticle settling and bioavailability. Here, the effects of an environmental strain of Pseudomonas aeruginosa on TiO2 nanoparticle agglomerates formed in aqueous media are described. Environmental scanning electron microscopy and cryogenic scanning electron microscopy visually demonstrated bacterial dispersion of large agglomerates formed in cell culture medium and in marsh water. For experiments in cell culture medium, quantitative image analysis verified that the degrees of conversion of large agglomerates into small nanoparticle-cell combinations were similar for 12-h-growth and short-term cell contact experiments. Dispersion in cell growth medium was further characterized by size fractionation: for agglomerated TiO2 suspensions in the absence of cells, 81% by mass was retained on a 5-µm-pore-size filter, compared to only 24% retained for biotic treatments. Filtrate cell and agglomerate sizes were characterized by dynamic light scattering, revealing that the average bacterial cell size increased from 1.4 µm to 1.9 µm because of nano-TiO2 biosorption. High-magnification scanning electron micrographs showed that P. aeruginosa dispersed TiO2 agglomerates by preferential biosorption of nanoparticles onto cell surfaces. These results suggest a novel role for bacteria in the environmental transport of engineered nanoparticles, i.e., growth-independent, bacterially mediated size and mass alterations of TiO2 nanoparticle agglomerates.

Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy.

Bacterial biofilms, i.e. surface-associated cells covered in hydrated extracellular polymeric substances (EPS), are often studied with high-resolution electron microscopy (EM). However, conventional desiccation and high vacuum EM protocols collapse EPS matrices which, in turn, deform biofilm appearances. Alternatively, wet-mode environmental scanning electron microscopy (ESEM) is performed under a moderate vacuum and without biofilm drying. If completely untreated, however, EPS is not electron dense and thus is not resolved well in ESEM. Therefore, this study was towards adapting several conventional SEM staining protocols for improved resolution of biofilms and EPS using ESEM. Three different biofilm types were used: 1) Pseudomonas aeruginosa unsaturated biofilms cultured on membranes, 2) P. aeruginosa cultured in moist sand, and 3) mixed community biofilms cultured on substrates in an estuary. Working with the first specimen type, a staining protocol using ruthenium red, glutaraldehyde, osmium tetroxide and lysine was optimized for best topographic resolution. A quantitative image analysis tool that maps relief, newly adopted here for studying biofilms, was used to compare micrographs. When the optimized staining and ESEM protocols were applied to moist sand cultures and aquatic biofilms, the smoothening effect that bacterial biofilms have on rough sand, and the roughening that aquatic biofilms impart on initially smooth coupons, were each quantifiable. This study thus provides transferable staining and ESEM imaging protocols suitable for a wide range of biofilms, plus a novel tool for quantifying biofilm image data.