Soot and other products of incomplete combustion play an important role in the chemistry of the atmosphere. As particles are exposed to trace gases, such as ozone, their chemistry and physical properties can be altered leading to changes in their optical properties, as well as their cloud condensation nuclei and ice nucleation abilities. These alterations can lead to changes in the global radiative budget and cloud microphysical processes, which in turn affect the climate. In this study, the chemical and physical changes associated with the oxidation of pyrene, anthracene, and carbon (lampblack) by ozone were investigated. Fourier Transform Infrared Spectroscopy was used to identify oxidation products and track reaction progress for these representative aerosols. A C=O band attributed to a carboxylic acid formation was observed for all three substances, at each level of exposure to ozone - 20 ppm, 40 ppm, and 80 ppm. Second order reaction rate constants ranged from 9.58 x 10-16 cm2 molecules-1 s-1 to 7.71 x 10-13 cm2 molecules-1 s-1. Measurements of water uptake, ice nucleation efficiency, and optical properties were obtained to determine whether any physical changes associated with the oxidation process occurred. Optical measurements show an increase in the ultra-violet absorption of anthracene, but not for pyrene, while an increase in the visible absorption for pyrene was observed, but not for anthracene. Oxidized soot froze at a warmer temperature (-22.8 degrees C) then fresh soot (-25.6 degrees C), showing an increase in ice nucleation efficiency. Our data indicates that oxidation by ozone does alter the chemistry and physical properties of the substances study, leading to possible changes in how they interact with atmospheric processes.

Ice formation largely influences the properties of clouds and hence it has an important impact on weather and climate. Primary ice formation is a consequence of either homogeneous or heterogeneous ice nucleation. The latter process is catalyzed by a foreign substance called Ice Nucleating Particle (INP). Mineral dust particles were found to contribute to atmospheric INPs. Most types of mineral dust are ice active below -20 °C. In contrast, atmospheric observations indicate that immersion freezing as one of the most important heterogeneous ice nucleation processes can occur at temperatures higher than -15 °C. One possible explanation for cloud glaciation at high temperatures might be the presence of biological material (e.g. bacteria) inducing ice nucleation. Our fundamental process and even qualitative understanding concerning atmospheric heterogeneous ice nucleation is limited. In the framework of the present thesis, experimental and theoretical work was carried out to improve the basic understanding of the immersion freezing behavior of mineral dust and bacterial INPs. On the basis of model simulations immersion freezing experiments were designed at the Leipzig Aerosol Cloud Interaction Simulator (LACIS). The immersion freezing behavior of mineral dust and bacterial INPs was studied in dependence of temperature and particle surface area/number at LACIS. As a results of the present thesis, it was found that the immersion freezing behavior of kaolinite being a proxy of mineral dust INPs does not depend on the droplet volume, but on the particle surface area. The kaolinite particles investigated caused freezing below -30 °C. In contrast, Ice Nucleation Active (INA) protein complexes that are attributed to bacterial INPs were found to induce freezing at -7 °C. Furthermore, it was shown that the ice nucleation activity of protein complexes is very similar regardless of whether the INA protein complex is attached to the outer cell membrane of intact bacteria or to cell membrane fragments. The immersion freezing ability depends on the number and type of INA protein complexes present in the droplet ensemble. The immersion freezing ability of mineral dust and bacterial INPs was parameterized accounting for the time effect. With this, results from literature could be reproduced for both INP types. These parameterizations can be used in e.g. cloud resolving atmospheric models.

Pseudomonas syringae produz núcleos de gelo biológicos de grande eficiência. Bioaerossóis destas células tem potencial de participar na glaciação de nuvens, podendo influenciar a precipitação. Foram estudadas como as condições as quais P. syringae está sujeita em suspensão na atmosfera afetam sua sobrevivência e sua atividade de nucleação de gelo. Duas cepas foram testadas, e ambas apresentaram baixa tolerância ao UV-C e ao UV-B, mas exibiram uma maior resistência quando expostas a um espectro semelhante ao encontrado no ambiente. A atividade de congelamento de uma das cepas (pv. syringae) não foi afetada pelo UV, enquanto que para a outra (pv. garcae) houve uma redução moderada. Em resposta à dessecação, pv. garcae foi substancialmente mais resistente que pv. syringae. Isto também afetou a nucleação de gelo das cepas. Em ensaios adicionais, estas bactérias foram expostas em um voo de balão estratosférico, e a uma simulação em laboratório das condições no topo da troposfera. Nestes dois experimentos, sobreviventes protegidos do UV foram recuperados. / Pseudomonas syringae produces biological ice nuclei of great efficiency. Bioaerosols of these cells have the potential to take part in cloud glaciation, possibly influencing the precipitation. It was studied how the conditions to which P. syringae is subjected while in suspension in the atmosphere affect its survival and its ice nucleation activity. Two strains were tested, and both showed a low tolerance to UV-C and UV-B, but exhibited a higher resistance when exposed to a spectrum similar to the one found in the environment. The freezing activity of one of the strains (pv. syringae) was not affected by the UV, while that for the other (pv. garcae) there was a moderate reduction. In response to desiccation, pv. garcae was substantially more resistant than pv. syringae. This also affected the ice nucleation by the strains. In additional assays, these bacteria were exposed in a stratospheric balloon flight, and to a laboratory simulation of the conditions at the top of the troposphere. After these two experiments, survivors protected from the UV were recovered.

No / We address the problem of recognition and growth of ice nuclei in simulation of supercooled bulk water. Bond orientation order parameters based on the spherical harmonics analysis are shown to be ineffective when applied to ice nucleation. Here we present an alternative method which robustly differentiates between hexagonal and cubic ice forms. The method is based on accumulation of the maximum projection of bond orientations onto a set of predetermined vectors, where different terms can contribute with opposite signs with the result that the irrelevant or incompatible molecular arrangements are damped out. We also introduce an effective cluster size by assigning a quality weight to each molecule in an ice-like cluster. We employ our cluster analysis in Monte Carlo simulation of homogeneous ice formation. Replica-exchange umbrella sampling is used for biasing the growth of the largest cluster and calculating the associated free energy barrier. Our results suggest that the ice formation can be seen as a two-stage process. Initially, short tetrahedrally arranged threads and rings are present; these become correlated and form a diffuse ice-genic network. Later, hydrogen bond arrangements within the amorphous ice-like structure gradually settle down and simultaneously `tune-up¿ nearby water molecules. As a result, a well-shaped ice core emerges and spreads throughout the system. The process is very slow and diverse owing to the rough energetic landscape and sluggish molecular motion in supercooled water, while large configurational fluctuations are needed for crystallization to occur. In the small systems studied so far the highly cooperative molecular rearrangements eventually lead to a relatively fast percolation of the forming ice structure through the periodic boundaries, which inevitably affects the simulation results. / EPSRC

No description available.

Fungi play important roles in various environments. Some of them infect plants and cause economically important diseases. However, many fungal pathogens cause similar symptoms or are even spread asymptomatically, making it difficult to identify them morphologically. Therefore, culture-independent, sequence-based diagnostic methods that can detect and identify fungi independently of the symptoms that they cause are desirable. Whole genome metagenomic sequencing has the potential to enable rapid diagnosis of plant diseases without culturing pathogens and designing pathogen-specific probes. In my study, the MinION nanopore sequencer, a portable single‐molecule sequencing platform developed by Oxford Nanopore Technologies, was employed to detect the fungus Calonectria pseudonaviculata (Cps), the causal agent of the devastating boxwood blight disease of the popular ornamental boxwood (Buxus spp.). Various DNA extraction methods and computational tools were compared. Detection was sensitive with an extremely low false positive rate for most methods. Therefore, metagenomic sequencing is a promising technology that could be implemented in routine diagnostics of fungal diseases. Other fungi may play important roles in the atmosphere because of their ice nucleation activity (INA). INA is the capacity of some particles to induce ice formation above the temperature that pure water freezes (-38°C). Importantly, INPs affect the ratio of ice crystals to liquid droplets in clouds, which in turn affects Earth's radiation balance and the intensity and frequency of precipitation. A few fungal species can produce ice nucleating particles (INPs) that cause ice formation at temperatures ≥ –10°C and they may be present in clouds. Two such fungal genera are Fusarium and Mortierella but little is known about their INPs and the genetic basis of their INA. In my study, F. avenaceum and M. alpina were examined in detail. INPs of both species were characterized and it was found that strains within both species varied in regards to the strength of INA. Whole genome sequencing and comparative genomic studies were then performed to identify putative INA genes. Differential expression analyses at different growth temperatures were also performed. INP properties of the two species shared similarities, both appearing to consist of secreted aggregates larger than 30 kDa. Low temperatures induced INA in both species. Lists of candidate INA genes were identified based on their presence in the strains with the strongest INA and/or induction of their expression at low temperatures and because they either encode secreted proteins or enzymes that produce other molecules known to have INA in other organisms. These genes can now be characterized further to help identify the fungal INA genes in both species. This can be expected to help increase our understanding of the role of fungal INA in the atmosphere. / Doctor of Philosophy / Fungi are important to life on Earth and play roles in the environments that surround us. On the one hand, fungi can make plants sick and some plant diseases may even cause economic losses to farmers. If the cause of a disease can be identified accurately in an early stage before symptoms develop, disease transmission may be prevented and plants may be protected from disease. However, it is a challenge to find out which fungus causes which disease since symptoms of different fungal diseases look very similar. Typically, we have to wait for plants to become very sick or we have to isolate the fungus that causes a disease to identity it, which may be time-consuming and not lead to precise identification. DNA sequencing technologies have the potential to lead to more sensitive, faster, and more accurate disease diagnosis and, therefore, may help prevent disease outbreaks. In my study, the MinION nanopore sequencer, a small portable device, was used to detect the fungus causing boxwood blight on boxwood. By loading the DNA of unhealthy boxwood on the device, the boxwood blight pathogen was identified within a very short time. Thus, this method is a promising diagnostic method that may be applied to detect other plant fungal diseases as well. On the other hand, fungi may affect Earth's climate by affecting how many water droplets in clouds are frozen, which in turn affects Earth's temperature and how often and how much it rains and snows. Fungi may affect the freezing of water droplets in clouds since some of them have ice nucleation activity (INA), which is the capacity to catalyze ice formation at a higher temperature than the temperature at which pure water freezes (-38°C), and they may be present in clouds. So far, INA has only been found in a few fungi, including the species Fusarium avenaceum and Mortierella alpina, but the mechanism of their INA is poorly understood. In my study, multiple F. avenaceum and M. alpina strains were examined in detail. Two approaches were used. First, strains in each species were compared with each other to find out how strong their INA is. Once it was found that they differed in their strength of INA, their genomes were sequenced and compared to find genes present in the most active strains and missing from the least active strains since it is these genes that may contribute to INA. It was also found that both fungal species had stronger INA when they were grown at lower temperatures. Therefore, the expression of their genes between higher and lower temperatures was compared to find the genes that were more highly expressed at lower temperatures since it is these genes that may cause INA. Based on previous studies, fungal INPs may either consist of secreted proteins or be the products of biosynthetic gene clusters. Therefore, the list of potential genes was reduced by looking for genes encoding either secreted proteins or biosynthetic gene clusters. The list of these potential INA genes will make it easier to identify the INA genes in F. avenaceum and M. alpina and determine the role of fungi in affecting the weather and climate on Earth.

Bacteria are ubiquitously present in every ecosystem on earth. While bacterial communities that reside in specific habitats, called the microbiota, have characteristic compositions, their constituents are exchanged between habitats. To understand the assembly processes and function of a microbial community in an ecosystem, it is thus important to identify its putative sources and sinks. The sources and sinks of the plant leaf microbiome, also called the phyllosphere microbiome, are still under debate. Here, I hypothesized that precipitation is a so far neglected source of the phyllosphere microbiome. Using 16S rRNA amplicon and metagenomic sequencing, I identified the genera Massilia, Sphingomonas, Methylobacterium, Pseudomonas, Acidiphilium, and Pantoea as members of the core rain microbiome in Blacksburg, VA. Further, I used rainwater as a bacterial inoculum to treat tomato plants. I showed that rain-borne bacteria of the genera Chryseobacterium, Enterobacter, Pantoea, Paenibacillus, Duganella, Streptomyces, Massilia, Shinella, Janthinobacterium, Erwinia, and Hyphomicrobium were significantly more abundant in the tomato phyllosphere 7 days post-inoculation, suggesting that these rain-borne bacteria successfully colonized the tomato phyllosphere and had a direct impact on the composition of its microbiome. These results were confirmed by comparing the phyllosphere microbiota of tomato plants grown under greenhouse conditions, and thus never exposed to rain, compared to plants grown outside under environmental conditions, including precipitation. Since a large diversity of bacteria is associated with rain, I also hypothesized that rain-borne bacteria are well adapted to environmental stresses, similar to the stressors microbial biopesticides are exposed to in the field. I thus explored rain as a source of resilient biopesticides to control fire blight, caused by the bacterial pathogen Erwinia amylovora, on apple. In an in-vitro dual culture assay, I identified rain-borne isolates displaying broad-range inhibition against E. amylovora and several other plant pathogens. Two rain-borne isolates, identified as Pantoea agglomerans and P. ananatis, showed the strongest inhibition of E. amylovora. Further experiments showed that these two Pantoea isolates survive under environmental conditions and have a strong protective effect against E. amylovora. However, protection from disease in an orchard was inconsistent, suggesting that the timing of application and formulations must be improved for field applications. Using a UV-mutagenesis screen and whole-genome sequencing, I found that a phenazine antibiotic produced by the P. agglomerans isolate was the likely active molecule that inhibited E. amylovora. Bacterial communities are constantly released as aerosols into the atmosphere from plant, soil, and aquatic sources. When in the atmosphere, bacteria may play crucial roles in geochemical processes, including the formation of precipitation. To understand the potential role of decaying vegetation as a source of atmospheric Ice Nucleation Particles (INPs), I analyzed a historic leaf litter sample collected in 1970 that had maintained Ice Nucleation Activity (INA) for 48 years. A culture-dependent analysis identified the bacterial species Pantoea ananatis and the fungal species Mortierella alpina to have INA and to be present in the leaf litter sample. Further, I determined that both P. ananatis and M. alpina produced heat-sensitive sub-micron INPs that may contribute to atmospheric INPs. The development of new sequencing technologies has facilitated our understanding of microbial community composition, assembly, and function. Most research in bacterial community composition is based on the sequencing of a single region of the 16S rRNA gene. Here, I tested the potential of culture-independent 16S rRNA sequencing of the phyllosphere microbiome for disease diagnosis. I compared the community composition of the microbiome of the aerial parts of cheddar pinks (Dianthus gratianopolitanus) that showed disease symptoms with the microbiome of healthy plants to identify the causative agent. However, I found that the pathogen is probably ubiquitous on cheddar pinks since it was present at similar abundance levels in symptomatic as well as healthy plants. Moreover, the low-resolution of 16S rRNA sequencing did not allow to identify the pathogen at the species or strain level. In summary, in this thesis, I found support for the hypothesis that rain is one of the sources of the phyllosphere microbiome, that rain is a promising source of biopesticides to control plant diseases in the field, that leaf litter is a source of atmospheric INPs, and that 16S rRNA sequencing is not well suited for pathogen identification in support of plant disease diagnosis. Finally, in additional research to which I contributed but that is not included in this thesis, I found that metagenomic sequencing can identify pathogens at the species and strain level and can overcome the limitations of 16S rRNA sequencing. / Doctor of Philosophy / Bacteria are present in nearly every ecosystem on earth. Bacterial communities that reside in a specific habitat are known as microbiota and have characteristic compositions and functions that directly impact the health of ecosystems. Microbiota associated with plants, the so-called plant microbiota, play a crucial role in plant fitness. Thus, it is important to study the assembly and diversity of plant microbiota and their impact on the ecosystem. The sources of leaf microbiota remain to be elucidated. Here, I have studied the contribution of rainfall to the bacteria that live on and in plant leaves. First, using DNA sequencing, I identified the bacteria present in rainfall in Blacksburg, VA. Then, using rain as bacterial inoculum, I found that some rain-borne bacteria, including members of the genera Pantoea, Massilia, Janthinobacterium, and Enterobacter, are efficient colonizers of tomato leaves. Either absence or low abundance of rain-borne bacteria from tomato leaves never exposed to rainfall confirmed further that bacteria in rain contribute to the assembly of plant leaf microbiota. The identification of all putative sources and sinks of leaf microbiota is important when trying to manipulate them to improve plant health and crop yield. Since I found that rainfall contains many different bacteria, I also studied the potential application of rain-borne bacteria in agriculture. The main limitations of commercial bio-pesticides are their poor survival and limited efficacy in the field. Here, I speculated that rain-borne bacteria are well adapted to environmental stressors and could represent efficient bio-pesticides under field conditions. In fact, I isolated two rain-borne bacteria from the genus Pantoea that strongly inhibited Erwinia amylovora, the causal agent of the fire blight disease of apple, in the laboratory under controlled conditions. However, I observed inconsistent results in a 2-year field trial in an orchard. Using mutagenesis and DNA sequencing, I found the active molecule that likely inhibited E. amylovora, in one of the rain-borne isolates. Finally, the access to newer and cheaper sequencing technologies has recently facilitated the study of bacteria at large scale. Most research of microbiota is based on the sequencing of a single region of one gene, the 16S rRNA gene. Here, I tested the potential of 16S rRNA sequencing of leaf microbiota for disease diagnosis. However, I identified the pathogen in healthy and diseased plants, suggesting its ubiquitous presence. Further, due to the low-resolution of 16S rRNA sequencing, it was impossible to identify the pathogen at the species level. In summary, I found that rain is a source that contributes to leaf microbiota, that rain is a promising source of bio-pesticides to control plant diseases, and that 16S rRNA sequencing is not recommended as a tool to diagnose plant diseases.

The mechanisms responsible for ice propagation on surfaces undergoing dropwise condensation have been determined and characterized. Based on experimental data acquired non-invasively with high speed quantitative microscopy, the freezing process was determined to occur by two distinct mechanisms: inter-droplet and intradroplet ice crystal growth. The inter-droplet crystal growth mechanism was responsible for the propagation of the ice phase between droplets while the intra-droplet crystal growth mechanism was responsible for the propagation of ice within individual droplets. The larger scale manifestation of these two mechanisms cooperating in tandem was designated as the aggregate freezing process. The dynamics of the aggregate freezing process were characterized in terms of the substrate thermal di usivity, the substrate temperature, the free stream air humidity ratio, and the interfacial substrate properties of roughness and contact angle, which were combined into a single surface energy parameter. Results showed that for a given thermal di usivity, the aggregate freezing velocity increased asymptotically towards a constant value with decreasing surface temperature, increasing humidity, and decreasing surface energy. The inter-droplet freezing velocity was found to be independent of substrate temperature and only slightly dependent on humidity and surface energy. The intra-droplet freezing velocity was determined to be a strong function of substrate temperature, a weaker function of surface energy, and independent of humidity. From the data, a set of correlational models were developed to predict the three freezing velocities in terms of the independent variables. These models predicted the majority of the measured aggregate, inter- and intra-droplet freezing velocities to within 15%, 10%, and 35%, respectively. Basic thermodynamic analyses of the inter- and intra-droplet freezing mechanisms showed that the dynamics of these processes were consistent with the kinetics of crystal growth from the vapor and supercooled liquid phases, respectively. The aggregate freezing process was also analyzed in terms of its constituent mechanisms; those results suggested that the distribution of liquid condensate on the surface has the largest impact on the aggregate freezing dynamics.

Ice formation largely influences the properties of clouds and hence it has an important impact on weather and climate. Primary ice formation is a consequence of either homogeneous or heterogeneous ice nucleation. The latter process is catalyzed by a foreign substance called Ice Nucleating Particle (INP). Mineral dust particles were found to contribute to atmospheric INPs. Most types of mineral dust are ice active below -20 °C. In contrast, atmospheric observations indicate that immersion freezing as one of the most important heterogeneous ice nucleation processes can occur at temperatures higher than -15 °C. One possible explanation for cloud glaciation at high temperatures might be the presence of biological material (e.g. bacteria) inducing ice nucleation. Our fundamental process and even qualitative understanding concerning atmospheric heterogeneous ice nucleation is limited. In the framework of the present thesis, experimental and theoretical work was carried out to improve the basic understanding of the immersion freezing behavior of mineral dust and bacterial INPs. On the basis of model simulations immersion freezing experiments were designed at the Leipzig Aerosol Cloud Interaction Simulator (LACIS). The immersion freezing behavior of mineral dust and bacterial INPs was studied in dependence of temperature and particle surface area/number at LACIS. As a results of the present thesis, it was found that the immersion freezing behavior of kaolinite being a proxy of mineral dust INPs does not depend on the droplet volume, but on the particle surface area. The kaolinite particles investigated caused freezing below -30 °C. In contrast, Ice Nucleation Active (INA) protein complexes that are attributed to bacterial INPs were found to induce freezing at -7 °C. Furthermore, it was shown that the ice nucleation activity of protein complexes is very similar regardless of whether the INA protein complex is attached to the outer cell membrane of intact bacteria or to cell membrane fragments. The immersion freezing ability depends on the number and type of INA protein complexes present in the droplet ensemble. The immersion freezing ability of mineral dust and bacterial INPs was parameterized accounting for the time effect. With this, results from literature could be reproduced for both INP types. These parameterizations can be used in e.g. cloud resolving atmospheric models.

Aufgrund ihrer speziellen Eigenschaften können sogenannte eisnukleierende Partikel die Bildung von Eis in Wolken katalysieren. Laboruntersuchungen zum Gefrierverhalten dieser Partikel haben sich als wertvoll erwiesen, wenn es um das Verständnis zugrunde liegender Prinzipien und Mechanismen geht. Eine Spezies, die in früheren Untersuchungen vernachlässigt wurde, ist Flugasche aus Kohleverbrennung. Kohle-Flugasche (KFA) wird aufgrund ineffizienter Filterung submikroner Partikel über die Schornsteine von Kraftwerken emittiert und kann, in Abhängigkeit der meteorologischen Bedingungen, die Vereisung von Wolken in der Nähe der Quelle und darüber hinaus beeinflussen. In dieser Arbeit wurde das Immersionsgefrierverhalten, d.h. der Einfluss eingeschlossener Partikel auf das Gefrieren unterkühlter Tropfen, für vier verschiedene KFA-Proben aus deutschen Kohlekraftwerken untersucht. Dabei wurden einerseits Tropfen untersucht, die ein einzelnes submikrones Partikel enthielten. Andererseits wurde das Gefrierverhalten von Suspensionstropfen, die eine Vielzahl verschieden großer Partikel beinhalteten, quantifiziert. Zusätzlich wurden die Proben, sowohl in ihrer Gesamtheit als auch in Form einzelner submikroner Partikel, bezüglich ihrer chemischen Zusammensetzung, Morphologie und Kristallographie analysiert. Es wurde festgestellt, dass die Gefriereffizienz der Proben innerhalb von Minuten abnimmt, sobald diese in Berührung mit Wasser kommen. Immersionsgefriermessungen mit purem Anhydrit (CaSO4 ), das in den Proben nachgewiesen wurde, zeigten einen ähnlichen Trend, d.h. eine abnehmende Effizienz mit zunehmender Suspensionszeit. Diese Beobachtung, und die Übereinstimmung von Messungen mit KFA-Suspensionspartikeln und Gips (CaSO 4 * 2H2O, ein Hydrat des Anhydrits), weisen darauf hin, dass Hydratation die Ursache für die Abnahme der Gefriereffizienz sein könnte. Dieser Einfluss von Probeneigenschaften und Methodologie auf das Immersionsgefrierverhalten von KFA-Partikeln muss bei der Abschätzung der Relevanz der Partikel für die atmosphärische Eisnukleation unbedingt berücksichtigt werden.:1. Introduction 2. Fundamentals 2.1 Ice nucleation theory 2.2 Properties of CFA particles 3. Materials and Methods 3.1 Materials 3.2 Methods 4. Results 4.1 Physicochemical sample characterization 4.2 Immersion freezing behavior of CFA 5. Discussion 5.1 Comparison to literature results 5.2 Physicochemical particle properties and immersion freezing behavior 5.3 Atmospheric implications 6. Summary and Conclusions 7. Outlook / Due to their specific properties, atmospheric ice-nucleating particles are able to catalyze ice formation in clouds. Laboratory studies investigating the freezing behavior of these particles have proven to be of unmatched value when attempting to understand underlying principles and mechanisms. One species that has almost entirely been neglected in previous ice nucleation studies is fly ash from coal combustion (CFA: coal fly ash). Emitted through the stacks of power plants due to inefficient filtering of submicron particles, CFA has the potential to influence cloud glaciation in source regions and beyond, depending on the meteorological conditions. In this thesis, the immersion freezing behavior, i.e., the influence of particles immersed in supercooled cloud droplets on ice nucleation, of four samples from German power plants was determined with the help of several single particle and bulk instruments. In parallel, single particles and bulk CFA were investigated with respect to their chemical composition, morphology, and crystallography. It was found that the immersion freezing efficiency of the CFA particles decreases in contact with water on the time scale of minutes. Hydration products, that were found in both single particles and in the bulk after suspension, could be responsible for this unique behavior. Immersion freezing measurements with pure anhydrite (anhydrous CaSO4 ), which is known to occur at the surface of CFA particles, showed the same qualitative trend, i.e., a decreasing efficiency with increasing suspension time. This observation, and the agreement between measurements with suspended CFA particles and gypsum (CaSO4 * 2H2O, a hydrate of anhydrite), support the hypothesis that hydration causes the observed decrease in immersion freezing efficiency. This influence of sample properties and methodology on the immersion freezing behavior of CFA must be taken into consideration when assessing the relevance of these particles for atmospheric ice nucleation.:1. Introduction 2. Fundamentals 2.1 Ice nucleation theory 2.2 Properties of CFA particles 3. Materials and Methods 3.1 Materials 3.2 Methods 4. Results 4.1 Physicochemical sample characterization 4.2 Immersion freezing behavior of CFA 5. Discussion 5.1 Comparison to literature results 5.2 Physicochemical particle properties and immersion freezing behavior 5.3 Atmospheric implications 6. Summary and Conclusions 7. Outlook