Engineered Nanomaterial Interactions with Bacterial Cells

University of Minnesota Ph.D. dissertation. May 2016. Major: Chemistry. Advisor: Christy Haynes. 1 computer file (PDF); xxx, 325 pages.Nanomaterials occur naturally in a variety of forms. They exist, for example, in the aerosols produced from sea spray and in the particulates produced from incomplete combustion of hydrocarbons. In the latter 20th century, development of instruments such as the scanning tunneling microscope and atomic force microscope have allowed us to directly see and to manipulate nanoscale matter. Armed with these instrumental capabilities and a desire to push the limits of our ability to create and manipulate matter, we have begun to engineer nanomaterials for our own use. Today, nanomaterials are used as additives in numerous commercial products to improve performance and/or reduce cost. Examples include silver nanomaterials in fabrics to inhibit microbial growth and titanium dioxide nanomaterials in outdoor paints to reduce weathering. Less often, nanomaterials serve a primary function in product performance; one important example of this is the use of nanoscale mixed metal oxides as cathode materials in lithium-ion batteries, used in some electric vehicles. The increasing commercial use of engineered nanomaterials increases direct human contact with nanoscale matter beyond that which formerly occurred naturally. Taking a proactive view of these developments, a small group of researchers began, in the early 2000s, to assess the implications of nanomaterial exposure on human health, giving rise to the field of nanotoxicology. In recent years, the field has expanded its focus beyond human health to include environmental health, recognizing that the waste streams resulting from the production, use, and disposal of products containing nanomaterials serve as new sources in natural environments. The goal of environmental nanotoxicity research, of which my dissertation research is a part, is to promote the sustainable use of engineered nanomaterials by assessing their environmental toxicity and informing their design in order to minimize environmental impact. As a project rooted in chemistry, my dissertation focuses in particular on identifying molecular structures, both nanomaterial and biological, that can be used to predict and control the environmental impact of nanomaterials. My research focuses on characterizing the interactions of commercially relevant nanomaterials with microorganisms, which play fundamental roles in healthy ecosystems. The bacterium Shewanella oneidensis MR-1, grown in culture, was used throughout my research as a model, albeit greatly simplified, of microorganism communities in natural environments. This particular bacterium was chosen due to the worldwide distribution of its genus, Shewanella, and its ability to survive in many environments, including aerobic, anaerobic, low-temperature, and high-salinity environments. Using this drastically simplified model greatly facilitates isolation of experimental variables, which would be much more difficult to achieve in the extremely chemically complex environment of soil or water samples collected from nature. This, in turn, greatly facilitates hypothesis testing. However, experimentation using samples obtained directly from nature is also necessary to develop a complete understanding of nanomaterial behavior in the environment. My research specifically addresses the following questions: What impact does natural organic matter (a ubiquitous component of natural sediments, soils, and water bodies) have on nanoparticle toxicity to bacteria in aquatic environments? How can we visually observe nanomaterial interactions with bacteria, both of which are near or below the diffraction limit of light, under hydrated conditions? Which structures on the bacterial cell surface primarily interact with nanomaterials? By what mechanism(s) might nanoscale battery cathode materials be toxic to bacteria, and how can we design less-toxic materials? The five major outcomes of my research, briefly summarized below, are presented in detail in Chapters 2-6. To address the first question (Chapters 2 and 3), I investigated the interactions between silver nanoparticles (also silver ions -- produced under aerobic conditions by the dissolution of silver nanoparticles) and natural organic matter. Natural organic matter is a complex mixture of polysaccharides, proteins, nucleic acids, and lipids and is produced through the decomposition of vegetative and microbial matter. Engineered nanoparticles entering natural environments, including soils, sediments, and water bodies, will inevitably encounter natural organic matter. Previous research has demonstrated that nanoparticle transport, persistence, and toxicity are influenced by interactions with natural organic matter. However, some reports conflict with these results and have demonstrated little or no impact of natural organic matter on nanoparticle behavior (e.g., colloidal stability). This conflict may result from a lack of attention paid to differences in the chemical composition of natural organic matter derived from various natural sources. The chemical heterogeneity of natural organic matter in various natural environments is significant, but researchers have often considered it to be a standard “class” of molecules that has common patterns of interaction with nanoparticles. My research, conducted in collaboration with Drs. Philippe Bühlmann and Maral Mousavi at the University of Minnesota—Twin Cities, sought to more specifically define the characteristics of natural organic matter that influence the behavior of silver nanoparticles and ions in natural aquatic environments. This research revealed that natural organic matter adsorption to silver nanoparticles and binding to silver ions depend greatly on the concentration of sites with high affinity for silver (e.g., sites rich in S and N). This result was affirmed by subsequent experiments with Shewanella, wherein silver nanoparticles and ions were less toxic only when first exposed to natural organic matter with this high binding affinity. This research also demonstrated a novel application of ion-selective electrodes in real-time monitoring of the dissolution kinetics of silver nanoparticles and the kinetics of natural organic matter binding to silver ions. This approach represents a significant improvement over the previous state-of-the-art (i.e., inductively-coupled plasma optical emission spectroscopy/mass spectrometry), which was limited to observing total silver concentration only (rather than distinguishing complexed and free forms of silver) and could be applied only at discrete time-points rather than being used for continuous measurements. To address the problem of visually observing nanomaterial interactions with bacterial cells (Chapter 4), I developed a novel and facile method to fluorescently stain bacterial cell surfaces for super-resolution fluorescence microscopy (SRFM). SRFM is uniquely capable of visualizing biological samples with high (sub-diffraction-limited) resolution under hydrated conditions. Electron microscopy, the current gold standard for high-resolution imaging, achieves higher resolution than SRFM but requires that samples be dehydrated and embedded in resin, procedures that can significantly alter the sample from its native state. Despite this advantage over electron microscopy, SRFM has been underutilized due to the complex fluorescent labeling strategies required. Current strategies based on genetic encoding of fluorescent proteins and fluorescent small-molecule labels require significant development time and are not generalizable across bacterial types (i.e., gram-positive and gram-negative bacteria). The fluorescent labeling strategy I developed uses only commercially available reagents and can be used to label both gram-positive and gram-negative bacterial cells. Utilizing the imaging instrumentation and resources at the Pacific Northwest National Laboratory, Richland, WA and with the collaboration of Dr. Galya Orr’s laboratory, super-resolution images of the gram-negative Shewanella oneidensis and the gram-positive Bacillus subtilis were acquired using two SRFM techniques (structured-illumination microscopy and stochastic optical reconstruction microscopy). In addition, structured-illumination microscopy was performed to visualize Shewanella oneidensis exposed to fluorescent cadmium selenide/zinc sulfide core-shell quantum dots under hydrated conditions. This method achieved sufficient resolution to determine that quantum dots were bound to the cell surface without translocating across the cell membrane. Research to further characterize the site of bacterial cell-nanomaterial interactions was motivated in part by the aforementioned SRFM imaging of Shewanella oneidensis exposed to quantum dots. My goal was to determine which surface membrane species mediated the interaction of the quantum dots with the bacterial cells. I hypothesized that lipopolysaccharides, abundant molecules in the outer leaflet of gram-negative bacterial cell membranes and extending from the membrane surface into the surrounding solution, was the critical species. Lipopolysaccharides form a highly cross-linked, hydrated barrier that helps protect the lipid membrane from damage caused by antimicrobial peptides, hydrophobic antibiotics, and surfactants. Using ethylenediaminetetraacetic acid to release divalent cation crosslinkers between adjacent molecules, I reduced the concentration of lipopolysaccharides in the outer membrane of live Shewanella oneidensis cells. After exposing cells with either intact or depleted lipopolysaccharides to gold nanoparticles, I quantified nanoparticle-to-cell association using a novel flow cytometry method developed in this work. This method exploited the high light-scattering cross section of gold nanoparticles as well as fluorescent labeling of cells to rapidly screen cells for gold nanoparticle association with high throughput. To more precisely assess lipopolysaccharide-nanoparticle interactions, parallel experiments using supported lipid bilayers were conducted by Dr. Kurt Jacobson in the laboratory of Dr. Joel Pedersen at the University of Wisconsin—Madison. The association between gold nanoparticles and supported lipid bilayers containing lipopolysaccharides was quantified using quartz crystal microbalance with dissipation. Use of supported lipid bilayers enabled greater control over lipopolysaccharide concentration and length than was possible using whole cells. Our combined results showed that lipopolysaccharide density and length determine the extent and distance of nanoparticle interaction with the gram-negative bacterial cell outer membrane. This work provides a basis for predicting the extent of interaction between nanoparticles and gram-negative bacteria, whose constituent lipopolysaccharides vary in length and density, and for engineering nanoparticles with enhanced or reduced bactericidal activity. The environmental implications of nanomaterial use in lithium-ion batteries is the subject of the final experimental chapter of my thesis, Chapter 6. This research, performed in collaboration with Mimi Hang from the laboratory of Dr. Robert Hamers at the University of Wisconsin—Madison, focused on nanoscale lithium nickel manganese cobalt oxide (NMC), currently used as a cathode material in the batteries of some commercially available electric vehicles. The goal of this research was to characterize the impact of NMC exposure on Shewanella oneidensis and to use this knowledge to propose a modified material design that reduces potential biological and environmental impacts. Our results show that exposure to NMC reduces bacterial growth and respiration and that this effect is attributable to leaching of metal ions (in particular Ni and Co species) from NMC in aqueous environments. Subsequently, we synthesized a series of Mn-enriched (and Ni- and Co-depleted) NMC species and characterized their impact on Shewanella oneidensis. Manganese enrichment significantly reduced but did not eliminate NMC’s toxicity. Ongoing research is focused on developing new synthetic strategies to limit metal ion leaching, including capping NMC with an insoluble layer, such as lithium iron phosphate. In summary, this research has identified several molecular-level phenomena that govern engineered nanomaterial interactions with bacterial cells, which are key members of natural ecosystems. By contributing to a more complete and fundamental understanding of engineered nanomaterial behavior in the environment, the author hopes this research will promote the sustainable and responsible use of engineered nanomaterials

Managing Wicked Herbicide-Resistance: Lessons from the Field

Herbicide resistance is ‘wicked’ in nature; therefore, results of the many educational efforts to encourage diversification of weed control practices in the United States have been mixed. It is clear that we do not sufficiently understand the totality of the grassroots obstacles, concerns, challenges, and specific solutions needed for varied crop production systems. Weed management issues and solutions vary with such variables as management styles, regions, cropping systems, and available or affordable technologies. Therefore, to help the weed science community better understand the needs and ideas of those directly dealing with herbicide resistance, seven half-day regional listening sessions were held across the United States between December 2016 and April 2017 with groups of diverse stakeholders on the issues and potential solutions for herbicide resistance management. The major goals of the sessions were to gain an understanding of stakeholders and their goals and concerns related to herbicide resistance management, to become familiar with regional differences, and to identify decision maker needs to address herbicide resistance. The messages shared by listening-session participants could be summarized by six themes: we need new herbicides; there is no need for more regulation; there is a need for more education, especially for others who were not present; diversity is hard; the agricultural economy makes it difficult to make changes; and we are aware of herbicide resistance but are managing it. The authors concluded that more work is needed to bring a community-wide, interdisciplinary approach to understanding the complexity of managing weeds within the context of the whole farm operation and for communicating the need to address herbicide resistance

Managing Herbicide Resistance: Listening to the Perspectives of Practitioners. Procedures for Conducting Listening Sessions and an Evaluation of the Process

Seven half-day regional listening sessions were held between December 2016 and April 2017 with groups of diverse stakeholders on the issues and potential solutions for herbicide-resistance management. The objective of the listening sessions was to connect with stakeholders and hear their challenges and recommendations for addressing herbicide resistance. The coordinating team hired Strategic Conservation Solutions, LLC, to facilitate all the sessions. They and the coordinating team used in-person meetings, teleconferences, and email to communicate and coordinate the activities leading up to each regional listening session. The agenda was the same across all sessions and included small-group discussions followed by reporting to the full group for discussion. The planning process was the same across all the sessions, although the selection of venue, time of day, and stakeholder participants differed to accommodate the differences among regions. The listening-session format required a great deal of work and flexibility on the part of the coordinating team and regional coordinators. Overall, the participant evaluations from the sessions were positive, with participants expressing appreciation that they were asked for their thoughts on the subject of herbicide resistance. This paper details the methods and processes used to conduct these regional listening sessions and provides an assessment of the strengths and limitations of those processes

Managing Wicked Herbicide-Resistance: Lessons from the Field

Herbicide resistance is ‘wicked’ in nature; therefore, results of the many educational efforts to encourage diversification of weed control practices in the United States have been mixed. It is clear that we do not sufficiently understand the totality of the grassroots obstacles, concerns, challenges, and specific solutions needed for varied crop production systems. Weed management issues and solutions vary with such variables as management styles, regions, cropping systems, and available or affordable technologies. Therefore, to help the weed science community better understand the needs and ideas of those directly dealing with herbicide resistance, seven half-day regional listening sessions were held across the United States between December 2016 and April 2017 with groups of diverse stakeholders on the issues and potential solutions for herbicide resistance management. The major goals of the sessions were to gain an understanding of stakeholders and their goals and concerns related to herbicide resistance management, to become familiar with regional differences, and to identify decision maker needs to address herbicide resistance. The messages shared by listening-session participants could be summarized by six themes: we need new herbicides; there is no need for more regulation; there is a need for more education, especially for others who were not present; diversity is hard; the agricultural economy makes it difficult to make changes; and we are aware of herbicide resistance but are managing it. The authors concluded that more work is needed to bring a community-wide, interdisciplinary approach to understanding the complexity of managing weeds within the context of the whole farm operation and for communicating the need to address herbicide resistance

Prospective evaluation of late-night salivary cortisol and cortisone by EIA and LC-MS/MS in suspected Cushing syndrome

Context: Late-night salivary cortisol (LNSC) measured by enzyme immunoassay (EIA-F) is a first-line screening test for Cushing syndrome (CS) with a reported sensitivity and specificity of \u3e90%. However, liquid chromatography-tandem mass spectrometry, validated to measure salivary cortisol (LCMS-F) and cortisone (LCMS-E), has been proposed to be superior diagnostically. Objective Setting and Main Outcome Measures: Prospectively evaluate the diagnostic performance of EIA-F, LCMS-F, and LCMS-E in 1453 consecutive late-night saliva samples from 705 patients with suspected CS. Design: Patients grouped by the presence or absence of at least one elevated salivary steroid result and then subdivided by diagnosis. Results: We identified 283 patients with at least one elevated salivary result; 45 had an established diagnosis of neoplastic hypercortisolism (CS) for which EIA-F had a very high sensitivity (97.5%). LCMS-F and LCMS-E had lower sensitivity but higher specificity than EIA-F. EIA-F had poor sensitivity (31.3%) for adrenocorticotropic hormone (ACTH)-independent CS (5 patients with at least 1 and 11 without any elevated salivary result). In patients with Cushing disease (CD), most nonelevated LCMS-F results were in patients with persistent/recurrent CD; their EIA-F levels were lower than in patients with newly diagnosed CD. Conclusions: Since the majority of patients with ≥1 elevated late-night salivary cortisol or cortisone result did not have CS, a single elevated level has poor specificity and positive predictive value. LNSC measured by EIA is a sensitive test for ACTH-dependent Cushing syndrome but not for ACTH-independent CS. We suggest that neither LCMS-F nor LCMS-E improves the sensitivity of late-night EIA-F for CS

Impact of TiO₂ Nanoparticles on Growth, Biofilm Formation, and Flavin Secretion in <i>Shewanella oneidensis</i>

Understanding of nanoparticle impacts on critical bacteria functions allows us to gain a mechanistic understanding of toxicity and guides us toward design rules for creating safe nanomaterials. Herein, biofilm formation, a general bacteria function, and riboflavin secretion, a species-specific function, were monitored in <i>Shewanella oneidensis</i>, a metal reducing bacterium, following exposure to a variety of TiO₂ nanoparticle types (synthesized, Aeroxide P25, and T-Eco). Transmission electron microscopy (TEM) images show that dosed nanoparticles are in close proximity to the bacteria, but they are not internalized. Using quartz crystal microbalance (QCM), it was revealed that <i>S. oneidensis</i> biofilm formation is slowed in the presence of nanoparticles. Though <i>S. oneidensis</i> grows more slowly in the presence of TiO₂ nanoparticles, riboflavin secretion, a function related to the <i>S. oneidensis</i> metal reducing capacity, was increased significantly in a nanoparticle dose-dependent manner. Both changes in biofilm formation and riboflavin secretion are supported by changes in gene expression in nanoparticle-exposed <i>S. oneidensis</i>. This broad study of bacterial nanotoxicity, including use of sensitive analytical tools for functional assessments of biofilm formation, riboflavin secretion, and gene expression, has implications for total ecosystem health as the use of engineered nanoparticles grows

Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes

Design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations and assessment of the potential implications of nanoparticle release into the environment requires understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate the electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the outer leaflet-solution interface. By manipulating the lipopolysaccharide content in <i>Shewanella oneidensis</i> outer membranes, we observed the electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) and second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. The association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long <i>O</i>-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides