Browsing by Subject "Silver"

Silver nanoparticles (AgNPs) are increasingly being integrated into a wide range of consumer products, such as air filters, washing machines, and textiles, due to their antimicrobial properties [1]. However, despite the beneficial applications of AgNPs into consumer products, it is likely that their use will facilitate the release of AgNPs into wastewater treatment plants, thereby possibly negatively impacting key microorganisms involved in nutrient removal. For this reason, it is important to characterize the effects of AgNPs in natural and engineered systems and to measure the antimicrobial effect of AgNPs on wastewater microorganisms. Polyvinyl alcohol coated AgNPs have already been linked to decreased nitrifying activity [2] and it is important to determine if AgNPs coated with other materials follow similar trends. Furthermore, it is likely that, with repeated exposure to AgNPs microbial communities could evolve and develop resistance to silver. Thus, a long-term effect of silver nanoparticle exposure could be a reduction of the efficacy of such products in a similar fashion to the development of microbial antibiotic resistance [3]. Therefore, it is critical that the impacts of these materials be ascertained in wastewater treatment systems to prevent long-term negative effects.

The objectives of this dissertation were to: 1) characterize the effect of several different AgNPs on the ammonia oxidizing bacterium (AOB) Nitrosomonas europaea and investigate possible mechanisms for toxicity, 2) test the effects of consumer product AgNPs on a wide range of heterotrophic bacteria, 3) evaluate the effects of AgNPs on bench scale wastewater sequencing batch reactors, and lastly 4) assess the impacts on microbial communities that are applied with AgNP spiked wastewater biosolids.

First, Nitrosomonas europaea was was selected because wastewater nitrifying microorganisms carry out the first step in nitrification and are known to be sensitive to a wide range of toxicants [4].The antimicrobial effects of AgNPs on the AOB N. europaea were measured by comparing nitrite production rates in a dose response assay and analyzing cell viability using the LIVE/DEAD® fluorescent staining assay. AgNP toxicity to N. europaea appeared to be largely nanoparticle coating dependent. While PVP coated AgNPs have shown reductions up to 15% in nitrite production at 20 ppm, other AgNPs such as gum arabic (GA) coated showed the same level of inhibition at concentrations of 2 ppm. The first mechanism of inhibition appears to be a post-transcriptional interference of AMO/HAO by either dissolved Ag or ROS, in treatments where membranes are not completely disrupted but nitrite production decreased (2 ppm GA AgNP and 2 ppm PVP AgNP treatments). The disruption of nitrification is dependent on AgNP characteristics, such as zeta potential and coating, which will dictate how fast the AgNP will release Ag+ and ROS production Finally, total membrane loss and release of internal cellular matter occur.

In order to test the effects of AgNP products available to consumers, simple bacterial toxicity tests were carried out on well-studied heterotrophic bacteria. A model gram-positive and gram-negative bacterium (B. subtilis and E. coli, respectively) was selected to assess any differences in sensitivity that may occur with the exposure to AgNPs. A third model gram-negative bacterium (P. aeruginosa) was chosen for its biofilm forming capabilities. In addition to testing pure nanoparticles, three silver supplements meant for ingestion, were randomly chosen to test with these three bacteria. Growth curve assays and LIVE/DEAD staining indicate that the consumer product AgNPs had the most significant inhibition on growth rates, but not membrane integrity. Overall, P. aeruginosa was most negatively affected by all AgNPs with nearly 100% growth inhibition for all 2 ppm AgNP treatments. TEM imaging also confirmed cell wall separation in P. aeruginosa and internal density differences for E. coli. The effects on B. subtilis, a gram-positive bacterium, were not as severe but toxicity was observed for several AgNPs at concentrations greater than 2 ppm. Citrate AgNPs appeared to have the most impact on membrane integrity, while other mechanisms such as internal thiol binding might have been at work for other AgNPs.

The effects of varying concentrations of pure AgNPs on complex microbial wastewater reactors are currently being tested. Eight bench-scale sequencing batch reactors were set up to follow the typical "fill, react, settle, decant, idle" method with an 8 hour hydraulic retention time and constant aeration. Reactors were fed synthetic wastewater and treatment efficiency is measured by monitoring effluent concentrations of COD, NH4+, and NO3-. The reactors were seeded with 500 mL of activated sludge from a local wastewater treatment plant. After reaching steady state, the reactors were spiked with 0.2 ppm gum arabic and citrate coated AgNPs. Treatment efficiency was monitored and results showed significant spikes and ammonia and COD immediately following the first spike, but the microbial community appeared to adapt for future AgNP spikes. Microbial community analysis (terminal restriction fragment length polymorphism) showed confirmed this hypothesis.

Overall, this dissertation asserts that by examining AgNP coating type, Ag+ dissolution rates and Stern layer surface charge, it may be possible to predict which AgNPs may be more detrimental wastewater treatment, but not all AgNPs will have the same effect. The results obtained herein must be expanded to other types of AgNPs and microorganisms of ecological importance.

Copper was first released into the environment as the result of the mass generation of oxygen from photosynthetic bacteria roughly 2.7 billion years ago. While it proved to be poisonous to early life on Earth, those that met the evolutionary challenge utilized the metal as a cofactor in enzymes to perform biochemically significant functions, while controlling intracellular levels of copper with a sophisticated network of trafficking proteins. Proteins and enzymes that utilize copper as a cofactor have evolved significantly different coordination environments than copper trafficking proteins, as a result of the different functions they perform. Of particular interest was characterizing the unique Cu(I) binding events observed in some of these proteins, the extracellular N-terminal regions of eukaryotic high affinity copper transport proteins (Ctr), and the bacterial periplamsic CusF protein of the CusFBCA Cu(I)/Ag(I) efflux pathway.

Model peptides corresponding to the methionine rich binding motifs (Mets motifs) were characterized in terms of Cu(I) binding affinity, stoichiometry, and metal specificity, via an ascorbic acid oxidation assay and electrospray ionization mass spectrometry. Metal induced structural features and coordination environments were elucidated with NMR, CD and X-ray spectroscopy. A series of peptides was also examined to infer the relative Cu(I) binding affinities, and susceptibility to oxidation, of methionine, histidine, cysteine residues found in copper binding motifs. The resistance of Cu+ specific peptides to metal catalyzed oxidation is also described. Attempts were also made to model the Cu(I)/Ag(I) tryptophan cation-π interaction observed in CusF.

Background: Silver nanoparticles (AgNPs) act as antimicrobials by releasing monovalent silver (Ag+) and are increasingly used in consumer products, thus elevating exposures in human and environmental populations. Materials and Methods: We evaluated Ag+ in a standard model of neuronal cell replication and differentiation, and then determined whether there were similar effects of the ion in vivo using zebrafish. Next, we compared Ag+ and AgNP exposures in the same two models and incorporated the effects of particle coating, size and composition. Conclusions: This work is the first to show that both Ag+ and AgNPs are developmental neurotoxicants in vitro and in vivo. Moreover, although both the soluble ion and the particles impair measures of neurodevelopment, the outcomes and underlying mechanisms of each toxicant are often wholly distinct. Superimposed on the dichotomies between Ag+ and AgNP exposures are clear effects of particle coating, size and composition that will necessitate evaluation of individual AgNP types when considering potential environmental and human health effects. The results presented here provide hazard identification that can help isolate the models and endpoints necessary for developing a risk assessment framework for the growing use of AgNPs.

Nanomaterials often exhibit enhanced reactivity relative to their larger colloidal counterparts because of the high specific surface area and number of imperfections on the crystal lattice at the nanoscale. Management of ecosystems, remediation of contaminated waters, and assessment of the potential risks from the industrial use on nanomaterials requires an understanding of the environmental factors that control the reactivity and bioavailability of natural and manufactured nanomaterials. Dissolved organic matter (DOM) acts as a moderator of reactivity and bioavailability for dissolved and particulate moieties in natural waters. DOM consists of a range of low and high molecular weight species that are complex and heterogeneous. It has been historically categorized based on operational definitions, rather than physical properties. In order to understand the effect of DOM on nanomaterials, there is an urgent need for information regarding specific properties of DOM, such as ligand groups.

The goal of this research was to study how cysteine, a low molecular weight metal-binding ligand, affects the composition and reactivity of nanoparticulate zinc sulfide and metallic silver. Zinc sulfide was used as a representative of nanoparticulate metal sulfide which occurs naturally in sulfidic environments. Metallic silver nanoparticles were also studied because of its wide use in consumer products. Both types of nanomaterials contain metal constituents (zinc and silver) that are expected to strongly bind to sulfhydryl-containing ligands (such as cysteine) in the environment. Serine is structurally similar to cysteine, with the only difference of a hydroxyl group in the place of the sulfhydryl group of cysteine. Therefore, serine was used for comparison as a hydroxyl-containing analogue to cysteine.

The aggregation kinetics of zinc and other metal sulfide nanoparticles in the presence of cysteine and serine were investigated using dynamic light scattering. Cysteine decreased aggregation rates of the particles, while serine had no effect on their aggregation behavior. Further experiments revealed that the mechanism of stabilization occurred through the adsorption of cysteine on zinc sulfide, which induced electrostatic charge on the particles surface. A direct link was established between the amount of cysteine sorbed and attachment efficiency, an indicator of the tendency of particles to aggregate. These results shed light on discrepancies in the literature between metal sulfide precipitation experiments conducted in our lab and work on the formation and aggregation of zinc sulfide nanoparticles on biofilms of sulfate reducing bacteria.

The early-stage growth and aggregation kinetics of zinc sulfide nanoclusters in the presence of cysteine was studied in detail using a suite of complementary techniques. Growth and aggregation experiments have been traditionally difficult to conduct due to instrumental precision issues, but newly developed analytical tools and software products have made it possible to study the early-stage formation of nanoclusters. Experiments with small angle X-ray scattering, X-ray diffraction, dynamic light scattering, and X-ray absorption spectroscopy at the extended fine structure range showed that cysteine controlled the growth and aggregation of zinc sulfide nanoclusters. The molar ratio between zinc, sulfide, and cysteine was a determining factor in the precipitation process. When zinc and sulfide were in equimolar concentrations with cysteine, very small nanoclusters of about 2.5 nm formed within 12 hours and aggregated to structures with hydrodynamic diameter larger than 100 nm. When cysteine was in excess of zinc and sulfide, aggregation was held to a minimum, but monomer nanoclusters were able to grow to about 5 nm in 12 hours. Overall, these results indicate the importance of thiol ligands on the monomer size, extent of aggregation, and aggregate structure of zinc sulfides.

The effect of metal ligands on metal bearing particle surfaces is of particular interest for manufactured nanoparticles, because they are typically coated with an organic coating during the production process. These coatings are sorbed on the particles surface and are likely to interfere between the metallic surface and the ligand. Dissolution experiments using citrate and polyvinylpyrrolidone (PVP) coated zero valent silver nanoparticles in the presence of cysteine and serine showed that cysteine dissolved both types of particles, while serine did not. Dissolution rates depended on the aggregation state of the particles exposed to cysteine. As indicated by zeta potential and adsorption measurements, cysteine replaced the coating on the particles surface and altered their aggregation pattern. X-ray absorption spectroscopy near the absorption edge showed partial oxidation of silver and formation of Ag(+I)-sulfur bonds, indicating that the thiol group in cysteine formed chemical bonds with oxidized surface silver atoms. A comparison between the two coatings showed that citrate coated particles dissolved approximately three times faster than PVP coated particles. Overall, these results show that metal binding ligands can drastically change the fate of manufactured silver nanoparticles in the environment and that this effect is moderated by surface coatings.

The results of this study suggest that cysteine, a metal binding ligand was able to induce and control transformations, such as growth, aggregation, dissolution, and surface reactivity of zinc sulfide and metallic silver nanoparticles. Cysteine adsorbed on metal sites on both ZnS and Ag particles, inducing changes on their surface charge. Aggregation of ZnS particles was slowed because of a net decrease in zeta potential compared to the bare particles. On the contrary, cysteine enhanced the aggregation of Ag particles, by replacing the citrate and PVP coatings on the particles surface. Finally, the cysteine-Ag(+I) bonds caused strong polarization on the particles surface and lead to the oxidative dissolution of the particles.

Overall, this research provides a better understanding of the fate of natural and manufactured nanoparticles in anaerobic waters, where thiols are present in significant amounts. It may also be used for risk assessment of manufactured nanomaterials and the production of safer and environmentally responsible materials.