Biological Nucleation Research Articles

Clothing Textiles as Carriers of Biological Ice Nucleation Active Particles.

Microplastics have littered the globe, with synthetic fibers being the largest source of atmospheric microplastics. Many atmospheric particles can act as ice nucleators, thereby affecting the microphysical and radiative properties of clouds and, hence, the radiative balance of the Earth. The present study focused on the ice-nucleating ability of fibers from clothing textiles (CTs), which are commonly shed from the normal wear of apparel items. Results from immersion ice nucleation experiments showed that CTs were effective ice nucleators active from -6 to -12 °C, similar to common biological ice nucleators. However, subsequent lysozyme and hydrogen peroxide digestion stripped the ice nucleation properties of CTs, indicating that ice nucleation was biological in origin. Microscopy confirmed the presence of biofilms (i.e., microbial cells attached to a surface and enclosed in an extracellular polysaccharide matrix) on CTs. If present in sufficient quantities in the atmosphere, biological particles (biofilms) attached to fibrous materials could contribute significantly to atmospheric ice nucleation.

Functional aggregation of cell-free proteins enables fungal ice nucleation.

Biological ice nucleation plays a key role in the survival of cold-adapted organisms. Several species of bacteria, fungi, and insects produce ice nucleators (INs) that enable ice formation at temperatures above -10 °C. Bacteria and fungi produce particularly potent INs that can promote water crystallization above -5 °C. Bacterial INs consist of extended protein units that aggregate to achieve superior functionality. Despite decades of research, the nature and identity of fungal INs remain elusive. Here, we combine ice nucleation measurements, physicochemical characterization, numerical modeling, and nucleation theory to shed light on the size and nature of the INs from the fungus Fusarium acuminatum. We find ice-binding and ice-shaping activity of Fusarium IN, suggesting a potential connection between ice growth promotion and inhibition. We demonstrate that fungal INs are composed of small 5.3 kDa protein subunits that assemble into ice-nucleating complexes that can contain more than 100 subunits. Fusarium INs retain high ice-nucleation activity even when only the ~12 kDa fraction of size-excluded proteins are initially present, suggesting robust pathways for their functional aggregation in cell-free aqueous environments. We conclude that the use of small proteins to build large assemblies is a common strategy among organisms to create potent biological INs.

Droplet freezing assays using a nanoliter osmometer

The ability to accurately record the temperature at which ice nucleation occurs is critical for studying biological ice nucleators. Several instruments have been designed and custom built to make such measurements, but they are not yet on the market. Here we reproducibly measure ice nucleation temperatures down close to the homogeneous nucleation temperature of −38 °C with a commercially available nanoliter osmometer, which we routinely use to assay the thermal hysteresis activity of antifreeze proteins. This instrument has both a wide operating temperature range and fine temperature control, while the oil immersion format on 12-well grids prevents droplet evaporation and surface nucleation events. The results obtained are consistent with those reported on other instruments in common use.

Lichen species across Alaska produce highly active and stable ice nucleators.

Forty years ago, lichens were identified as extraordinary biological ice nucleators (INs) that enable ice formation at temperatures close to 0°C. By employing INs, lichens thrive in freezing environments that surpass the physiological limits of other vegetation, thus making them the majority of vegetative biomass in northern ecosystems. Aerosolized lichen INs might further impact cloud glaciation and have the potential to alter atmospheric processes in a warming Arctic. Despite the ecological importance and formidable ice nucleation activities, the abundance, diversity, sources, and role of ice nucleation in lichens remain poorly understood. Here, we investigate the ice nucleation capabilities of lichens collected from various ecosystems across Alaska. We find ice-nucleating activity in lichen to be widespread, particularly in the coastal rainforest of Southeast Alaska. Across 29 investigated lichen, all species show ice nucleation temperatures above -15 °C and ~30% initiate freezing at temperatures above -6 °C. Concentration series of lichen ice nucleation assays in combination with statistical analysis reveal that the lichens contain two subpopulations of INs, similar to previous observations in bacteria. However, unlike the bacterial INs, the lichen INs appear as independent subpopulations resistant to freeze-thaw cycles and against temperature treatment. The ubiquity and high stability of the lichen INs suggest that they can impact local atmospheric processes and that ice nucleation activity is an essential trait for their survival in cold environments.

History of Discovery and Environmental Role of Ice Nucleating Bacteria.

The phenomenon of biological ice nucleation that is exhibited by a variety of bacteria is a fascinating phenotype, which has been shown to incite frost damage to frost-sensitive plants and has been proposed to contribute to atmospheric processes that affect the water cycle and earth's radiation balance. This review explores the several possible drivers for the evolutionary origin of the ice nucleation phenotype. These bacteria and the gene required for this phenotype have also been exploited in processes as diverse as reporter gene assays to assess environmentally responsive gene expression in various plant pathogenic and environmental bacteria and in the detection of foodborne human pathogens when coupled with host-specific bacteriophage, whereas ice nucleating bacteria themselves have been exploited in the production of artificial snow for recreation and oil exploration and in the process of freezing of various food products. This review also examines the historical development of our understanding of ice nucleating bacteria, details of the genetic determinants of ice nucleation, and features of the aggregates of membrane-bound ice nucleation protein necessary for catalyzing ice. Lastly, this review also explores the role of these bacteria in limiting the supercooling ability of plants and the strategies and limitations of avoiding plant frost damage by managing these bacterial populations by bactericides, antagonistic bacteria, or cultural control strategies.

Art’s Work in the Age of Biotechnology: Shaping Our Genetic Futures

Art’s Work in the Age of Biotechnology: Shaping Our Genetic Futures

Biological Ice Nucleators in Snow Samples from Greece

The formation of precipitation in clouds is initiated by inorganic and organic/biological ice nuclei. Certain species of bacteria and fungi are known to act as efficient biological ice nuclei at temperatures between −10 and 0 °C. Biological ice nuclei have been found and characterized in precipitation samples (snow, rain, and hail). We investigated the presence of warm temperature biological ice nuclei in 17 fresh snow samples from Greece and isolated and partially characterized ice nucleation active bacteria from these. All snow samples contained particles or other material active as ice nuclei at −9 °C in concentrations ranging from 3 to 943 nuclei/L. The numbers of this class of ice nuclei were reduced or eliminated after incubating snowmelt concentrates at 100 °C for 15 min and by treatment with lysozyme, a bacterial cell wall-degrading enzyme. These findings indicate the presence of microbial ice nuclei in snow samples from Greece. We also isolated ice nucleation active bacteria from some of the samples. These bacteria belong to genus Pseudomonas and are common on plants and soil. This is the first report on biological ice nuclei in precipitation samples from Greece.

Responses of the Plant Cell Wall to Sub-Zero Temperatures: A Brief Update.

Our general understanding of plant responses to sub-zero temperatures focuses on mechanisms that mitigate stress to the plasma membrane. The plant cell wall receives comparatively less attention, and questions surrounding its role in mitigating freezing injury remain unresolved. Despite recent molecular discoveries that provide insight into acclimation responses, the goal of reducing freezing injury in herbaceous and woody crops remains elusive. This is likely due to the complexity associated with adaptations to low temperatures. Understanding how leaf cell walls of herbaceous annuals promote tissue tolerance to ice does not necessarily lead to understanding how meristematic tissues are protected from freezing by tissue-level barriers formed by cell walls in overwintering tree buds. In this mini-review, we provide an overview of biological ice nucleation and explain how plants control the spatiotemporal location of ice formation. We discuss how sugars and pectin side chains alleviate adhesive injury that develops at sub-zero temperatures between the matrix polysaccharides and ice. The importance of site-specific cell-wall elasticity to promote tissue expansion for ice accommodation and control of porosity to impede ice growth and promote supercooling will be presented. How specific cold-induced proteins modify plant cell walls to mitigate freezing injury will also be discussed. The opinions presented in this report emphasize the importance of a plant’s developmental physiology when characterizing mechanisms of freezing survival.

Microbial ice nucleators scavenged from the atmosphere during simulated rain events

Rain and snow collected at ground level have been found to contain biological ice nucleators. These ice nucleators have been proposed to have originated in clouds, where they may have participated in the formation of precipitation via ice phase nucleation. We conducted a series of field experiments to test the hypothesis that at least some of the microbial ice nucleators (prokaryotes and eukaryotes) present in rain may not originate in clouds but instead be scavenged from the lower atmosphere by rainfall. Thirty-three simulated rain events were conducted over four months off the side of the Smart Road Bridge in Blacksburg, VA, USA. In each event, sterile water was dispensed over the side of the bridge and recovered in sterile containers in an open fallow agricultural field below (a distance of ∼55 m). Microbes scavenged from the simulated rain events were cultured and their ice nucleation activity was examined. Putative microbial ice nucleators were cultured from 94% (31/33) of the simulated rain events, and represented 1.5% (121/8331) of the total colonies assayed. Putative ice nucleators were subjected to additional droplet freezing assays, and those confirmed through these repeated assays represented 0.4% (34/8331) of the total. Mean CFUs scavenged by simulated rain ranged from 2 to 267 CFUs/mL. Scavenged ice nucleators belong to a number of taxa including the bacterial genera Pseudomonas, Pantoea, and Xanthomonas, and the fungal genera Fusarium, Humicola, and Mortierella. An ice-nucleating strain of the fungal genus Penicillium was also recovered from a volumetric air sampler at the study site. This work expands our knowledge of the scavenging properties of rainfall, and suggests that at least some ice nucleators in natural precipitation events may have been scrubbed from the atmosphere during rainfall, and thus are not likely to be involved in precipitation.

Effects of different temperature treatments on biological ice nuclei in snow samples

The heat tolerance of biological ice nucleation activity (INA) depends on their types. Different temperature treatments may cause varying degrees of inactivation on biological ice nuclei (IN) in precipitation samples. In this study, we measured IN concentration and bacterial INA in snow samples using a drop freezing assay, and compared the results for unheated snow and snow treated at 40 °C and 90 °C. At a measured temperature of −7 °C, the concentration of IN in untreated snow was 100–570 L−1, whereas the concentration in snow treated at 40 °C and 90 °C was 31–270 L−1 and 2.5–14 L−1, respectively. In the present study, heat sensitive IN inactivated by heating at 40 °C were predominant, and ranged 23–78% of IN at −7 °C compared with untreated samples. Ice nucleation active Pseudomonas strains were also isolated from the snow samples, and heating at 40 °C and 90 °C inactivated these microorganisms. Consequently, different temperature treatments induced varying degrees of inactivation on IN in snow samples. Differences in the concentration of IN across a range of treatment temperatures might reflect the abundance of different heat sensitive biological IN components.

Biological ice nucleation initiates hailstone formation

AbstractCloud condensation and ice nuclei in the troposphere are required precursors to cloud and precipitation formation, both of which influence the radiative balance of Earth. The initial stage of hailstone formation (i.e., the embryo) and the subsequent layered growth allow hail to be used as a model for the study of nucleation processes in precipitation. By virtue of the preserved particle and isotopic record captured by hailstones, they represent a unique form of precipitation that allows direct characterization of the particles present during atmospheric ice nucleation. Despite the ecological and economic consequences of hail storms, the dynamics of hailstone nucleation, and thus their formation, are not well understood. Our experiments show that hailstone embryos from three Rocky Mountain storms contained biological ice nuclei capable of freezing water at warm, subzero (°C) temperatures, indicating that biological particles can act as nucleation sites for hailstone formation. These results are corroborated by analysis of δD and δ18O from melted hailstone embryos, which show that the hailstones formed at similarly warm temperatures in situ. Low densities of ice nucleation active abiotic particles were also present in hailstone embryos, but their low concentration indicates they were not likely to have catalyzed ice formation at the warm temperatures determined from water stable isotope analysis. Our study provides new data on ice nucleation occurring at the bottom of clouds, an atmospheric region whose processes are critical to global climate models but which has challenged instrument‐based measurements.

Freezing nucleation apparatus puts new slant on study of biological ice nucleators in precipitation

Abstract. For decades, drop-freezing instruments have contributed to a better understanding of biological ice nucleation and its likely implications for cloud and precipitation development. Yet, current instruments have limitations. Drops analysed on a cold stage are subject to evaporation and potential contamination. The use of closed tubes provides a partial solution to these problems, but freezing events are still difficult to be clearly detected. Here, we present a new apparatus where freezing in closed tubes is detected automatically by a change in light transmission upon ice development, caused by the formation of air bubbles and crystal facets that scatter light. Risks of contamination and introduction of biases linked to detecting the freezing temperature of a sample are then minimized. To illustrate the performance of the new apparatus we show initial results of two assays with snow samples. In one, we repeatedly analysed the sample (208 tubes) over the course of a month with storage at +4 °C, during which evidence for biological ice nucleation activity emerged through an increase in the number of ice nucleators active around −4 °C. In the second assay, we indicate the possibility of increasingly isolating a single ice nucleator from a precipitation sample, potentially determining the nature of a particle responsible for a nucleation activity measured directly in the sample. These two seminal approaches highlight the relevance of this handy apparatus for providing new points of view in biological ice nucleation research.

Bioprecipitation: a feedback cycle linking Earth history, ecosystem dynamics and land use through biological ice nucleators in the atmosphere

Landscapes influence precipitation via the water vapor and energy fluxes they generate. Biologically active landscapes also generate aerosols containing microorganisms, some being capable of catalyzing ice formation and crystal growth in clouds at temperatures near 0°C. The resulting precipitation is beneficial for the growth of plants and microorganisms. Mounting evidence from observations and numerical simulations support the plausibility of a bioprecipitation feedback cycle involving vegetated landscapes and the microorganisms they host. Furthermore, the evolutionary history of ice nucleation-active bacteria such as Pseudomonas syringae supports that they have been part of this process on geological time scales since the emergence of land plants. Elucidation of bioprecipitation feedbacks involving landscapes and their microflora could contribute to appraising the impact that modified landscapes have on regional weather and biodiversity, and to avoiding inadvertent, negative consequences of landscape management.

High concentrations of biological aerosol particles and ice nuclei during and after rain

Abstract. Bioaerosols are relevant for public health and may play an important role in the climate system, but their atmospheric abundance, properties, and sources are not well understood. Here we show that the concentration of airborne biological particles in a North American forest ecosystem increases significantly during rain and that bioparticles are closely correlated with atmospheric ice nuclei (IN). The greatest increase of bioparticles and IN occurred in the size range of 2–6 μm, which is characteristic for bacterial aggregates and fungal spores. By DNA analysis we found high diversities of airborne bacteria and fungi, including groups containing human and plant pathogens (mildew, smut and rust fungi, molds, Enterobacteriaceae, Pseudomonadaceae). In addition to detecting known bacterial and fungal IN (Pseudomonas sp., Fusarium sporotrichioides), we discovered two species of IN-active fungi that were not previously known as biological ice nucleators (Isaria farinosa and Acremonium implicatum). Our findings suggest that atmospheric bioaerosols, IN, and rainfall are more tightly coupled than previously assumed.

Urediospores of rust fungi are ice nucleation active at > −10 °C and harbor ice nucleation active bacteria

Abstract. Various features of the biology of the rust fungi and of the epidemiology of the plant diseases they cause illustrate the important role of rainfall in their life history. Based on this insight we have characterized the ice nucleation activity (INA) of the aerially disseminated spores (urediospores) of this group of fungi. Urediospores of this obligate plant parasite were collected from natural infections of 7 species of weeds in France, from coffee in Brazil and from field and greenhouse-grown wheat in France, the USA, Turkey and Syria. Immersion freezing was used to determine freezing onset temperatures and the abundance of ice nuclei in suspensions of washed spores. Microbiological analyses of spores from France, the USA and Brazil, and subsequent tests of the ice nucleation activity of the bacteria associated with spores were deployed to quantify the contribution of bacteria to the ice nucleation activity of the spores. All samples of spores were ice nucleation active, having freezing onset temperatures as high as −4 °C. Spores in most of the samples carried cells of ice nucleation-active strains of the bacterium Pseudomonas syringae (at rates of less than 1 bacterial cell per 100 urediospores), but bacterial INA accounted for only a small fraction of the INA observed in spore suspensions. Changes in the INA of spore suspensions after treatment with lysozyme suggest that the INA of urediospores involves a polysaccharide. Based on data from the literature, we have estimated the concentrations of urediospores in air at cloud height and in rainfall. These quantities are very similar to those reported for other biological ice nucleators in these same substrates. However, at cloud level convective activity leads to widely varying concentrations of particles of surface origin, so that mean concentrations can underestimate their possible effects on clouds. We propose that spatial and temporal concentrations of biological ice nucleators active at temperatures > −10 °C and the specific conditions under which they can influence cloud glaciation need to be further evaluated so as to understand how evolutionary processes could have positively selected for INA.

Improvements to an Empirical Parameterization of Heterogeneous Ice Nucleation and Its Comparison with Observations

AbstractA framework for an empirical parameterization (EP) of heterogeneous nucleation of ice crystals by multiple species of aerosol material in clouds was proposed in a 2008 paper by the authors. The present paper reports improvements to specification of a few of its empirical parameters. These include temperatures for onset of freezing, baseline surface areas of aerosol observed in field campaigns over Colorado, and new parameters for properties of black carbon, such as surface hydrophilicity and organic coatings. The EP’s third group of ice nucleus (IN) aerosols is redefined as that of primary biological aerosol particles (PBAPs), replacing insoluble organic aerosols. A fourth group of IN is introduced—namely, soluble organic aerosols.The new EP predicts IN concentrations that agree well with aircraft data from selected traverses of shallow wave clouds observed in five flights (1, 3, 4, 6, and 12) of the 2007 Ice in Clouds Experiment–Layer Clouds (ICE-L). Selected traverses were confined to temperatures between about −25° and −29°C in layer cloud without homogeneously nucleated ice from aloft. Some of the wave clouds were affected by carbonaceous aerosols from biomass burning and by dust from dry lakebeds and elsewhere. The EP predicts a trend between number concentrations of heterogeneously nucleated ice crystals and apparent black carbon among the five wave clouds, observed by aircraft in ICE-L. It is predicted in terms of IN activity of black carbon.The EP’s predictions are consistent with laboratory and field observations not used in its construction, for black carbon, dust, primary biological aerosols, and soluble organics. The EP’s prediction of biological ice nucleation is validated using coincident field observations of PBAP IN and PBAPs in Colorado.

Distribution of Ice Nucleation-Active (INA) Bacteria from Rain-water and Air

Certain bacteria that induce biological ice nucleation are suspected to play an important role in condensation and ice nuclei formation in clouds. Those bacteria can produce biological ice nucleator 1 , which is a protein and usually found on leaf surface and air. Most studies on INA bacteria were conducted in subtropical areas. In this study, INA bacteria were isolated from rain-water and air between March to May 2008 from Jakarta, Bogor, Bekasi, Tangerang, and Depok. The percentage of INA bacteria from rainfall for those area are 19.4, 18.7, 5.3, 2.2, and 6.4% respectively, while percentage for air are 9.5, 6.5, 0, 2.7, and 1.8%. The highest incidence of INA bacteria were from rain-water and air found in sample from Jakarta and then followed by the samples from Bogor. It was shown that the percentage of INA bacteria from rain-water was higher than air for all of the samples from different areas. The isolate from Jakarta (isolate A 32 ) which had the highest activity for ice nucleation, with the temperature classification at −2.7 °C, revealed 100% similarity with Pantoea sp. The presence of INA bacteria in rain-water and air might play an important role in nucleation process which is required for rainfall induction.

Effects of sulfate coatings on the ice nucleation properties of a biological ice nucleus and several types of minerals

An optical microscope coupled to a flow cell was used to study the ice nucleation properties of uncoated and coated mineral dust and SNOMAX (a proxy for biological ice nucleators made from cells of Pseudomonas syringae) at temperatures ranging from 234 to 247 K. We define the onset conditions as the relative humidity (RH) and temperature at which the first ice nucleation event was observed. The results show that H2SO4 coatings modified the ice nucleation properties of all the minerals studied. For kaolinite and illite, the acid coatings increased the RH over ice (RHi) required for ice nucleation by ∼30% RHi; for montmorillonite and quartz, the acid coatings increased the RHi by ∼20% RHi. NH4HSO4 coatings also influenced the ice nucleation properties of kaolinite particles. In addition, our results indicate that SNOMAX is a reasonably good ice nucleus, having onset values between 110 to 120% RHi. In contrast to the mineral studies, sulfuric acid coatings did not hinder the ice nucleating ability of SNOMAX particles. Combined, our results support the idea that anthropogenic emissions of SO2 and NH3 may influence the ice‐nucleating properties of mineral dust particles. From our laboratory data, we also determined contact angles (θ) between the heterogeneous nuclei and ice embryos according to classical nucleation theory to parameterize the laboratory data for inclusion in atmospheric models. The data show that for uncoated ice nuclei the contact angles are small (below ∼20°), but for mineral particles coated with sulfuric acid, the contact angles are larger (above ∼60°).

Geographic, seasonal, and precipitation chemistry influence on the abundance and activity of biological ice nucleators in rain and snow

Biological ice nucleators (IN) function as catalysts for freezing at relatively warm temperatures (warmer than -10 degrees C). We examined the concentration (per volume of liquid) and nature of IN in precipitation collected from Montana and Louisiana, the Alps and Pyrenees (France), Ross Island (Antarctica), and Yukon (Canada). The temperature of detectable ice-nucleating activity for more than half of the samples was > or = -5 degrees C based on immersion freezing testing. Digestion of the samples with lysozyme (i.e., to hydrolyze bacterial cell walls) led to reductions in the frequency of freezing (0-100%); heat treatment greatly reduced (95% average) or completely eliminated ice nucleation at the measured conditions in every sample. These behaviors were consistent with the activity being bacterial and/or proteinaceous in origin. Statistical analysis revealed seasonal similarities between warm-temperature ice-nucleating activities in snow samples collected over 7 months in Montana. Multiple regression was used to construct models with biogeochemical data [major ions, total organic carbon (TOC), particle, and cell concentration] that were accurate in predicting the concentration of microbial cells and biological IN in precipitation based on the concentration of TOC, Ca(2+), and NH(4)(+), or TOC, cells, Ca(2+), NH(4)(+), K(+), PO(4)(3-), SO(4)(2-), Cl(-), and HCO(3)(-). Our results indicate that biological IN are ubiquitous in precipitation and that for some geographic locations the activity and concentration of these particles is related to the season and precipitation chemistry. Thus, our research suggests that biological IN are widespread in the atmosphere and may affect meteorological processes that lead to precipitation.

Ubiquity of Biological Ice Nucleators in Snowfall

Despite the integral role of ice nucleators (IN) in atmospheric processes leading to precipitation, their sources and distributions have not been well established. We examined IN in snowfall from mid- and high-latitude locations and found that the most active were biological in origin. Of the IN larger than 0.2 micrometer that were active at temperatures warmer than -7 degrees C, 69 to 100% were biological, and a substantial fraction were bacteria. Our results indicate that the biosphere is a source of highly active IN and suggest that these biological particles may affect the precipitation cycle and/or their own precipitation during atmospheric transport.