An optical microscope coupled to a flow cell was used to investigate ice nucleation on five atmospherically relevant mineral dusts at temperatures ranging from 233 to 247 K. Kaolinite and muscovite particles were found to be efficient ice nuclei in the deposition mode, requiring relative humidities with respect to ice (RHi) below 112% in order to initiate ice crystal formation. Quartz and calcite particles, by contrast, were poor ice nuclei, requiring relative humidities close to water saturation before ice crystals would form. Montmorillonite particles were efficient ice nuclei at temperatures below 241 K, but poor ice nuclei at higher temperatures. In several cases, there was a lack of quantitative agreement between these data and previously published work. This can be explained by several factors including mineral source, particle size, observation time and surface area available for nucleation. Heterogeneous nucleation rates (Jhet) were calculated from the onset data. Jhet values ranged from 60 to 1100 cm-²s-¹ for the five minerals studied. These values were then used to calculate contact angles (θ) for each mineral according to classical nucleation theory. The contact angles measured for kaolinite and muscovite ranged from 6 to 12º; for quartz and calcite the contact angles were much higher, ranging from 25 to 27º. The contact angles measured for montmorillonite were less than 15º at temperatures below 241 K, and above 20º at higher temperatures. The reported Jhet and θ values may allow for a more direct comparison between laboratory studies and can be used when modeling ice cloud formation in the atmosphere. The roles of H₂SO₄ and (NH4)₂SO₄ coatings on the ice nucleating properties of kaolinite were also investigated. Onset data was collected for H₂SO₄ coated and (NH4)₂SO₄ coated kaolinite particles at temperatures ranging from 233 to 247 K. In contrast to uncoated kaolinite particles, which were effective ice nuclei, H₂SO₄ coated particles were found to be poor ice nuclei, requiring relative humidities close to water saturation before nucleating ice at all temperatures studied. (NH4)₂SO₄ coated particles were poor ice nuclei at 245 K, but effective ice nuclei at 236 K.

An optical microscope coupled to a flow cell was used to investigate ice nucleation on five atmospherically relevant mineral dusts at temperatures ranging from 233 to 247 K. Kaolinite and muscovite particles were found to be efficient ice nuclei in the deposition mode, requiring relative humidities with respect to ice (RHi) below 112% in order to initiate ice crystal formation. Quartz and calcite particles, by contrast, were poor ice nuclei, requiring relative humidities close to water saturation before ice crystals would form. Montmorillonite particles were efficient ice nuclei at temperatures below 241 K, but poor ice nuclei at higher temperatures. In several cases, there was a lack of quantitative agreement between these data and previously published work. This can be explained by several factors including mineral source, particle size, observation time and surface area available for nucleation. Heterogeneous nucleation rates (Jhet) were calculated from the onset data. Jhet values ranged from 60 to 1100 cm-²s-¹ for the five minerals studied. These values were then used to calculate contact angles (θ) for each mineral according to classical nucleation theory. The contact angles measured for kaolinite and muscovite ranged from 6 to 12º; for quartz and calcite the contact angles were much higher, ranging from 25 to 27º. The contact angles measured for montmorillonite were less than 15º at temperatures below 241 K, and above 20º at higher temperatures. The reported Jhet and θ values may allow for a more direct comparison between laboratory studies and can be used when modeling ice cloud formation in the atmosphere. The roles of H₂SO₄ and (NH4)₂SO₄ coatings on the ice nucleating properties of kaolinite were also investigated. Onset data was collected for H₂SO₄ coated and (NH4)₂SO₄ coated kaolinite particles at temperatures ranging from 233 to 247 K. In contrast to uncoated kaolinite particles, which were effective ice nuclei, H₂SO₄ coated particles were found to be poor ice nuclei, requiring relative humidities close to water saturation before nucleating ice at all temperatures studied. (NH4)₂SO₄ coated particles were poor ice nuclei at 245 K, but effective ice nuclei at 236 K.

An optical microscope coupled to a flow cell was used to investigate ice nucleation on five atmospherically relevant mineral dusts at temperatures ranging from 233 to 247 K. Kaolinite and muscovite particles were found to be efficient ice nuclei in the deposition mode, requiring relative humidities with respect to ice (RHi) below 112% in order to initiate ice crystal formation. Quartz and calcite particles, by contrast, were poor ice nuclei, requiring relative humidities close to water saturation before ice crystals would form. Montmorillonite particles were efficient ice nuclei at temperatures below 241 K, but poor ice nuclei at higher temperatures. In several cases, there was a lack of quantitative agreement between these data and previously published work. This can be explained by several factors including mineral source, particle size, observation time and surface area available for nucleation. Heterogeneous nucleation rates (Jhet) were calculated from the onset data. Jhet values ranged from 60 to 1100 cm-²s-¹ for the five minerals studied. These values were then used to calculate contact angles (θ) for each mineral according to classical nucleation theory. The contact angles measured for kaolinite and muscovite ranged from 6 to 12º; for quartz and calcite the contact angles were much higher, ranging from 25 to 27º. The contact angles measured for montmorillonite were less than 15º at temperatures below 241 K, and above 20º at higher temperatures. The reported Jhet and θ values may allow for a more direct comparison between laboratory studies and can be used when modeling ice cloud formation in the atmosphere. The roles of H₂SO₄ and (NH4)₂SO₄ coatings on the ice nucleating properties of kaolinite were also investigated. Onset data was collected for H₂SO₄ coated and (NH4)₂SO₄ coated kaolinite particles at temperatures ranging from 233 to 247 K. In contrast to uncoated kaolinite particles, which were effective ice nuclei, H₂SO₄ coated particles were found to be poor ice nuclei, requiring relative humidities close to water saturation before nucleating ice at all temperatures studied. (NH4)₂SO₄ coated particles were poor ice nuclei at 245 K, but effective ice nuclei at 236 K. / Science, Faculty of / Chemistry, Department of / Graduate

Doctor of Philosophy / The temperature at which water freezes can be significantly increased by particles known as ice nucleating particles (INPs). The presence of these particles in the atmosphere plays an important role in determining weather patterns and climate by helping ice crystals to form. INPs can come from different sources, but biological organisms, especially microbes, are some of the most efficient producers. For decades, scientists have known about the ice-nucleating abilities of certain bacteria, but much of the research has focused on a specific gene found in a single group of bacteria. However, little is known about how other microbes can induce freezing. This study aims to fill this gap by characterizing INPs and exploring the genetics and regulation of microbial ice nucleation in one bacterium and several fungi. The goals of this research were threefold: (1) to create a detailed family tree of a group of bacteria called Pantoea, (2) to study how changes in a specific gene cluster in a bacterium called Lysinibacillus parviboronicapiens affect its ability to nucleate ice, and (3) to identify genes in fungi that may also be responsible for ice nucleation. To do this, I used advanced genetic analysis methods to compare the DNA sequences of different Pantoea species, studied gene expression in L. parviboronicapiens, and used computational tools to predict which genes in fungi (specifically in Fusarium and Mortierellaceae) might play a role in ice nucleation. vii One of the main findings was the creation of a new, more reliable family tree for Pantoea, based on 2,509 genes. This tree helps scientists better understand the relationships between different Pantoea species, allowing scientists to be able to identify and study them more easily and safely by determining which of them are pathogenic and which are harmless or even beneficial. In L. parviboronicapiens, changes in the gene cluster responsible for ice nucleation caused the bacteria to grow differently, and the study also uncovered a connection between ice nucleation and molecular transport across the bacterial cell wall. For the fungi, I identified several potential genes that may be involved in ice nucleation, and modeled their structures to better understand how they might work. This research provides new insights into the diversity of microbes that contribute to ice formation and advances our understanding of how they nucleate ice. These findings could improve how scientists model weather and climate and could have practical applications in biotechnology and environmental science.

Heterogeneous freezing processes in which atmospheric aerosols act as ice nuclei (IN) cause nucleation of ice crystals in the atmosphere. Heterogeneous nucleation can occur through several freezing mechanisms, including contact and immersion freezing. The mechanism by which this freezing occurs depends on the ambient conditions and composition of the IN. Aerosol properties change through chemical aging and reactions with atmospheric oxidants such as ozone. We have conducted a series of laboratory experiments using an optical microscope apparatus equipped with a cooling stage to determine how chemical oxidation changes the ability of atmospheric aerosols to act as IN. Freezing temperatures are reported for aerosols composed of fresh and oxidized soot and polyaromatic hydrocarbons (PAHs) including anthracene, phenanthrene, and pyrene. Our results show that oxidized soot particles initiate ice freezing events at significantly warmer temperatures than fresh soot, 3 °C on average. All oxidized PAHs studied had significantly warmer freezing temperatures than fresh samples. The chemical changes presumably causing the improved ice nucleation efficiency were observed using Fourier Transform Infrared Spectroscopy with Horizontal Attenuated Total Reflectance (FTIR-HATR). The addition of C=O bonds at the surface of the soot and PAHs led to changes in freezing temperatures. Finally, we have used classical nucleation theory to derive heterogeneous nucleation rates for the IN compositions in this research. The overall efficiency of the IN can be compared in order of least efficient to most efficient: fresh phenanthrene, fresh anthracene, fresh soot, oxidized phenanthrene, fresh pyrene, oxidized anthracene, oxidized soot, and oxidized pyrene. Overall oxidation of aerosols increases their ability to act as IN. Our results suggest that oxidation processes facilitate freezing at warmer temperatures at a broader range of conditions on the atmosphere.

Thesis (Ph.D.)--Ohio University, June, 2009. / Release of full electronic text on OhioLINK has been delayed until June 1, 2010. Includes bibliographical references (leave 107)

Thesis (Ph.D.)--Ohio University, June, 2009. / Title from PDF t.p. Release of full electronic text on OhioLINK has been delayed until June 1, 2010. Includes bibliographical references (leave 107)

Since the 1970s, a growing body of research has suggested that bacteria play an active role in precipitation. These bacteria are capable of catalyzing the formation of ice at relatively warm temperatures utilizing a specific protein family which aids in the binding of water molecules. However, the overall biodiversity, concentration, and relationship of ice nucleation active (ice+) bacteria with air mass trajectories and precipitation chemistry is not well studied. Precipitation events were collected over 15 months in Blacksburg, VA and ice+ bacteria were isolated from these samples. From these samples, 33,134 total isolates were screened for ice nucleation activity (INA) at -8 °C. A total of 593 of these isolated positively confirmed for INA at the same temperature in subsequent tests. The precipitation events had a mean concentration of 384±147 colony forming units per liter. While the majority of confirmed ice+ bacteria belonged to the gammaproteobacteria, a well-studied class of bacteria, including ice+ species of Pseudomonas, Pantoea, and Xanthomonas, two isolates were identified as Lysinibacillus, a Gram-positive member of the Firmicute phylum. These two isolates represent the first confirmed non-gammaproteobacteria with INA. After further characterization, the two isolates of Lysinibacillus did not appear to use a protein to freeze water. Instead, the Lysinibacillus isolates used a secreted, nanometer-sized molecule that is heat, lysozyme, and proteinase resistant. In an attempt to identify the mechanism responsible for this activity, species type strains were tested for INA and UV mutants were generated to knock out the ice+ phenotype. Based on these results, only members of the species L. parviboronicapiens exhibit INA and the genes responsible for the activity may lie within a type-1 polyketide synthase/non-ribosomal peptide synthase gene cluster. This gene cluster is absent from the genomes of all non-ice+ strains of Lysinibacillus, and contains mutations in five of the nine ice nucleation inactive mutants generated from the rain isolated strain. To better understand the phylogenetic relationship among ice+ Lysinibacillus, a comprehensive reference guide was compiled to provide the most up-to-date information regarding the genus and each of its species. This reference will be available to other researchers investigating Lysinibacillus species or other closely related genera. / PHD / It is a common misconception that water freezes at 0°C (32°F). In clouds, water may remain liquid until -37 to -40°C (-35 to -40°F). At temperatures warmer than this, water molecules must collect around small particles that can help form ice, called ice nuclei. Numerous ice nuclei have been identified, ranging from dirt and dust, to volcanic ash, and even to pollen, fungi, and bacteria. One of these bacteria, Pseudomonas syringae, was identified as an ice nucleus in the 1970’s when it was discovered that it was increasing susceptibility of corn to frost damage. Since then, other Pseudomonas species as well as other bacteria within the same family of bacteria have been shown to have the ability to freeze water at relatively warm temperatures utilizing a specialized protein. Despite numerous studies on how these bacteria can exist in the atmosphere and how they can freeze water, the extent of this freezing ability, the concentration of bacteria in precipitation, and how cloud chemistry affects these bacteria has not been widely studied. In this study, precipitation was collected over the course of 15 months and the bacteria found within the collected precipitation were checked to see if they could act as ice nuclei. We found many of the previously described bacterial ice nuclei in the precipitation samples, but also identified a previously unidentified bacterium capable of freezing water. This bacterium, Lysinibacillus parviboronicapiens, does not use the same method of freezing as the other described bacterial ice nuclei. As such, we set out to determine the method it uses. We have determined that this bacterium utilizes a heat-stable, nanometer-sized particle that is not a protein. To better understand this molecule, representative strains of each species of this genus of bacteria were tested for their ability to freeze water, however, only this species has the ability. To further identify the molecule, UV radiation was used to disrupt the bacteria’s ability to freeze water, and the genes responsible were identified. Based on these results, we have tentatively identified the responsible genes as part of a polyketide synthase gene cluster. This gene cluster is responsible for producing small molecules that provide some survival advantage for the bacteria, in our case, possibly the ability to freeze water. As a final step, and to help serve other researchers, a comprehensive analysis of the entire Lysinibacillus genus has been performed and a reference guide has been generated to help describe and distinguish individual species.

The indirect aerosol effect contributes to major uncertainties in determining the radiation budget of the earth. A large uncertainty is due to the formation of ice clouds onto natural or anthropogenic aerosols. Field studies have shown that mineral type particles are often associated with ice crystals in the mid-upper troposphere and given the long residence time in the atmosphere of dust particles (~2 weeks in the absence of precipitation), their contribution to ice formation processes is not fully defined. In order to probe ice formation onto natural mineral dust in a setting where it could be suspended as aerosol, a new continuous flow diffusion chamber (CFDC) was built. This allowed investigations of the effects of total aerosol surface area and particle size. The CFDC was also used in an international inter-comparison of ice nucleation instrumentation to compare efficiencies of soot, biological aerosol (bacteria) and samples of natural desert dusts from different regions of the world. The laboratory observations were parameterized using nucleation rates (Jhet) and contact angles () as described by classical nucleation theory. For both this experimental technique and a static one developed during the candidate’s Masters degree, mineral dust particulate proved to be the most efficient ice nuclei (IN) activating at RH with respect to ice (RHi) as low as 105% at T = 233 K. The efficiency varied with particle size and aerosol surface area (SA). Large particles or higher SA activated at lower RHi than small particles or lower SA. The static chamber was sensitive to the first ice event out of a large SA and therefore gave true onset RHi, which was lower than the onset defined by the CFDC studies, which was not sensitive to a single ice event. In addition the static chamber used a broader size range of particulate matter, including super micron particles while the CFDC particles sizes were restricted to below 0.5 µm. Soot and organic coated dust particles were inefficient IN compared to pure dust. Soot aerosols showed some efficiency at T < 233K where deposition ice formation was apparent. The hygroscopic organics had intermediate ice activity between dusts and alkyl-organics and soot. Bacteria aerosols were active in the deposition mode for T as high as 247 K. Contact angles () computed for ice germs forming onto natural mineral dust were small, 7<  < 29, at 223 K for RH ranging from ice to water saturation. It was concluded that there is no single value for the onset of ice formation in the atmosphere via deposition freezing. The associated contact angles show that there is a distribution of active sites on IN and that not all active sites have the same affinity for initiation of ice formation even within the same aerosol type. This work provides evidence that deposition mode nucleation can be an alternate pathway to homogeneous nucleation when mineral aerosols are present in the troposphere since the high T - low RH conditions required for deposition mode nucleation are more easily encountered in the atmosphere than the low T - high RH required for homogeneous nucleation.

Nucleation of ice crystals in the atmosphere often occurs through heterogeneous freezing processes facilitated by an atmospheric aerosol that acts as the ice nuclei (IN). Depending on ambient conditions and aerosol composition, heterogeneous nucleation will occur through one of several mechanisms including the contact and immersion freezing mechanisms. Through a series of contact freezing experiments, we have characterized the ability of aerosols composed of volcanic ash, soot, and peat soil, to act as ice nuclei (IN) as a function of temperature. The immersion freezing ability of the ash particles has also been measured. In these studies, an optical microscope apparatus equipped with a cooling stage and a digital camera was used to observe the freezing events. For each experiment, a particular IN was placed in contact with the surface, or immersed in the bulk, of an ultra pure water droplet. The droplet was then subjected to freezing-melting cycles resulting in 25 independent measurements of the freezing temperature of the droplet. In the volcanic ash experiments, we observed contact freezing at warmer temperatures than immersion freezing. As contact freezing IN, the peat was the most effective with an average contact freezing temperature of -10.5 �C, followed by volcanic ash (-11.2 �C), and then soot (-25.6 �C). In addition, we have used classical nucleation theory to identify the contact parameters and nucleation rates for the compositions explored.