Diversity and ice nucleation activity of Pseudomonas syringae in drone-based water samples from eight lakes in Austria.

Bacteria from the Pseudomonas syringae complex (comprised of at least 15 recognized species and more than 60 different pathovars of P. syringae sensu stricto) have been cultured from clouds, rain, snow, streams, rivers, and lakes. Some strains of P. syringae express an ice nucleation protein (hereafter referred to as ice+) that catalyzes the heterogeneous freezing of water. Though P. syringae has been sampled intensively from freshwater sources in the U.S. and France, little is known about the genetic diversity and ice nucleation activity of P. syringae in other parts of the world. We investigated the haplotype diversity and ice nucleation activity at -8 °C (ice+) of strains of P. syringae from water samples collected with drones in eight freshwater lakes in Austria. A phylogenetic analysis of citrate synthase (cts) sequences from 271 strains of bacteria isolated from a semi-selective medium for Pseudomonas revealed that 69% (188/271) belonged to the P. syringae complex and represented 32 haplotypes in phylogroups 1, 2, 7, 9, 10, 13, 14 and 15. Strains within the P. syringae complex were identified in all eight lakes, and seven lakes contained ice+ strains. Partial 16S rDNA sequences were analyzed from a total of 492 pure cultures of bacteria isolated from non-selective medium. Nearly half (43.5%; 214/492) were associated with the genus Pseudomonas. Five of the lakes (ALT, GRU, GOS, GOL, and WOR) were all distinguished by high levels of Pseudomanas (p ≤ 0.001). HIN, the highest elevation lake, had the highest percentage of ice+ strains. Our work highlights the potential for uncovering new haplotypes of P. syringae in aquatic habitats, and the use of robotic technologies to sample and characterize microbial life in remote settings.

Seasonal ice nucleation activity of water samples from alpine rivers and lakes in Obergurgl, Austria.

Heterogeneous ice nucleation plays an important role in many environmental processes such as ice cloud formation, freezing of water bodies or biological freeze protection in the cryosphere. New information is needed about the seasonal availability, nature, and activity of ice nucleating particles (INPs) in alpine environments. These INPs trigger the phase transition from liquid water to solid ice at elevated subzero temperatures. We collected water samples from a series of alpine rivers and lakes (two valleys and their rivers, an artificial pond, and a natural lake system) in Obergurgl, Austria in June 2016, July 2016, November 2016, and May 2017. Each alpine river and lake was sampled multiple times across different seasons, depending on site access during different times of the year. Water samples were filtered through a 0.22 µm membrane filter to separate microbial INPs from the water, and both fractions were analyzed for ice nucleation activity (INA) by an emulsion freezing method. Microorganisms were cultured from the filters, and the cultures then analyzed for INA. Portions of the filtered samples were concentrated by lyophilization to observe potential enhancement of INA. Two sediment samples were taken as reference points for inorganic INPs. Sub-micron INPs were observed in all of the alpine water sources studied, and a seasonal shift to a higher fraction of microbial ice nucleators cultured on selective media was observed during the winter collections. Particles larger than 0.22 µm showed INA, and microbes were cultured from this fraction. Results from 60 samples gave evidence of a seasonal change in INA, presence of submicrometer INPs, and show the abundance of culturable microorganisms, with late spring and early summer showing the most active biological INPs. With additional future research on this topic ski resorts could make use of such knowledge of geographical and seasonal trends of microbial INPs in freshwater habitats in order to improve the production of artificial snow.

Assessment of Artificial and Natural Transport Mechanisms of Ice Nucleating Particles in an Alpine Ski Resort in Obergurgl, Austria.

Artificial snow production is a crucial part of modern skiing resorts in Austria and globally, and will develop even more so with changing precipitation patterns and a warming climate trend. Producing artificial snow requires major investments in energy, water, infrastructure and manpower for skiing resorts. In addition to appropriate meteorological conditions, the efficiency of artificial snow production depends on heterogeneous ice-nucleation, which can occur at temperatures as high as -2°C when induced by specific bacterial ice nucleating particles (INPs). We aimed to investigate the presence, source and ice nucleating properties of these particles in the water cycle of an alpine ski resort in Obergurgl, Tyrol, Austria. We sampled artificial snow, river water, water pumped from a storage pond and compared it to samples collected from fresh natural snow and aged piste snow from the area. Particles from each sampled system were characterized in order to determine their transport mechanisms at a ski resort. We applied a physical droplet freezing assay [DRoplet Ice Nuclei Counter Zurich (DRINCZ)] to heated and unheated samples to characterize the biological and non-biological component of IN-activity. Bacterial abundance and community structure of the samples was obtained using quantitative PCR and Illumina Mi-Seq Amplicon Sequencing, and their chemical properties were determined by liquid ion-chromatography, energy dispersive X-ray spectroscopy (EDX) and scanning electron microscopy (SEM). The results show the flow of biological and inorganic material from the river to the slopes, an uptake of new microorganisms through the air and the piping, and possible proliferation or introduction of ice nucleation active biological particles in aged piste snow. Natural snow, as the first stage in this system, had the lowest amount of ice nucleation active particles and the least amount of biological and mineral particles in general, yet shares some microbial characteristics with fresh artificial snow.

Metastable Nitric Acid Trihydrate in Ice Clouds.

The composition of high-altitude ice clouds is still a matter of intense discussion. The constituents in question are ice and nitric acid hydrates, but the exact phase composition of clouds and its formation mechanisms are still unknown. In this work, conclusive evidence for a long-predicted phase, alpha-nitric acid trihydrate (alpha-NAT), is presented. This phase was characterized by a combination of X-ray and neutron diffraction experiments, allowing a convincing structure solution. Furthermore, vibrational spectra (infrared and inelastic neutron scattering) were recorded and compared with theoretical calculations. A strong interaction between water ice and alpha-NAT was found, which explains the experimental spectra and the phase-transition kinetics. On the basis of these results, we propose a new three-step mechanism for NAT formation in high-altitude ice clouds.

Does the emulsification procedure influence freezing and thawing of aqueous droplets?

Here we investigate the freezing and thawing properties of aqueous solutions in oil emulsions, with a particular focus on investigating the influence of the oil and surfactant and the stirring time of the emulsion. Specifically, we employ optical cryomicroscopy in combination with differential scanning calorimetry to study the phase behavior of emulsified 25 wt. % ammonium sulfate droplets in the temperature range down to 93 K. We conclude that the nucleation temperature does not vary with oil-surfactant combination, that is, homogeneous nucleation is probed. However, incomplete emulsification and non-unimodal size distribution of dispersed droplets very often result in heterogeneous nucleation. This in turn affects the distribution of freeze-concentrated solution and the concentration of the solid ice/ammonium sulfate mixture and, thus, the phase behavior at sub-freezing temperatures. For instance, the formation of letovicite at 183 K critically depends on whether the droplets have frozen heterogeneously or homogeneously. Hence, the emulsification technique can be a very strong technique, but it must be ensured that emulsification is complete, i.e., a unimodal size distribution of droplets near 15 µm has been reached. Furthermore, phase separation within the matrix itself or uptake of water from the air may impede the experiments.