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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 fungusFusarium acuminatum. We find ice-binding and ice-shaping activity ofFusariumIN, 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.FusariumINs 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.

 

Abstract. Forty years ago, lichens were identified as extraordinary biological icenucleators (INs) that enable ice formation at temperatures close to0 ∘C. By employing INs, lichens thrive in freezing environmentsthat surpass the physiological limits of other vegetation, thus making themthe majority of vegetative biomass in northern ecosystems. Aerosolizedlichen INs might further impact cloud glaciation and have the potential toalter atmospheric processes in a warming Arctic. Despite the ecologicalimportance and formidable ice nucleation activities, the abundance,diversity, sources, and role of ice nucleation in lichens remain poorlyunderstood. Here, we investigate the ice nucleation capabilities of lichenscollected from various ecosystems across Alaska. We find ice nucleatingactivity in lichen to be widespread, particularly in the coastal rainforestof southeast Alaska. Across 29 investigated lichen, all species show icenucleation temperatures above −15 ∘C, and ∼30 %initiate freezing at temperatures above −6 ∘C. Concentrationseries of lichen ice nucleation assays in combination with statisticalanalysis reveal that the lichens contain two subpopulations of INs, similarto previous observations in bacteria. However, unlike the bacterial INs, thelichen INs appear as independent subpopulations resistant to freeze–thawcycles and against temperature treatment. The ubiquity and high stability ofthe lichen INs suggest that they can impact local atmospheric processes andthat ice nucleation activity is an essential trait for their survival incold environments.

 

The appearance of ice I in the smallest possible clusters and the nature of its phase coexistence with liquid water could not thus far be unraveled. The experimental and theoretical infrared spectroscopic and free-energy results of this work show the emergence of the characteristic hydrogen-bonding pattern of ice I in clusters containing only around 90 water molecules. The onset of crystallization is accompanied by an increase of surface oscillator intensity with decreasing surface-to-volume ratio, a spectral indicator of nanoscale crystallinity of water. In the size range from 90 to 150 water molecules, we observe mixtures of largely crystalline and purely amorphous clusters. Our analysis suggests that the liquid–ice I transition in clusters loses its sharp 1st-order character at the end of the crystalline-size regime and occurs over a range of temperatures through heterophasic oscillations in time, a process without analog in bulk water. 

Ice nucleation in the atmosphere influences cloud properties, altering precipitation and the radiative balance, ultimately regulating Earth’s climate. An accepted ice nucleation pathway, known as deposition nucleation, assumes a direct transition of water from the vapor to the ice phase, without an intermediate liquid phase. However, studies have shown that nucleation occurs through a liquid phase in porous particles with narrow cracks or surface imperfections where the condensation of liquid below water saturation can occur, questioning the validity of deposition nucleation. We show that deposition nucleation cannot explain the strongly enhanced ice nucleation efficiency of porous compared with nonporous particles at temperatures below −40 °C and the absence of ice nucleation below water saturation at −35 °C. Using classical nucleation theory (CNT) and molecular dynamics simulations (MDS), we show that a network of closely spaced pores is necessary to overcome the barrier for macroscopic ice-crystal growth from narrow cylindrical pores. In the absence of pores, CNT predicts that the nucleation barrier is insurmountable, consistent with the absence of ice formation in MDS. Our results confirm that pore condensation and freezing (PCF), i.e., a mechanism of ice formation that proceeds via liquid water condensation in pores, is a dominant pathway for atmospheric ice nucleation below water saturation. We conclude that the ice nucleation activity of particles in the cirrus regime is determined by the porosity and wettability of pores. PCF represents a mechanism by which porous particles like dust could impact cloud radiative forcing and, thus, the climate via ice cloud formation.

 

Heterogeneous ice nucleation in the atmosphere regulates cloud properties, such as phase (ice versus liquid) and lifetime. Aerosol particles of marine origin are relevant ice nucleating particle sources when marine aerosol layers are lifted over mountainous terrain and in higher latitude ocean boundary layers, distant from terrestrial aerosol sources. Among many particle compositions associated with ice nucleation by sea spray aerosols are highly saturated fatty acids. Previous studies have not demonstrated their ability to freeze dilute water droplets. This study investigates ice nucleation by monolayers at the surface of supercooled droplets and as crystalline particles at temperatures exceeding the threshold for homogeneous freezing. Results show the poor efficiency of long chain fatty acid (C16, C18) monolayers in templating freezing of pure water droplets and seawater subphase to temperatures of at least −30 °C, consistent with theory. This contrasts with freezing of fatty alcohols (C22 used here) at nearly 20 °C warmer. Evaporation of μL-sized droplets to promote structural compression of a C19 acid monolayer did not favor warmer ice formation of drops. Heterogeneous ice nucleation occurred for nL-sized droplets condensed on 5 to 100 μm crystalline particles of fatty acid (C12 to C20) at a range of temperatures below −28 °C. These experiments suggest that fatty acids nucleate ice at warmer than −36 °C only when the crystalline phase is present. Rough estimates of ice active site densities are consistent with those of marine aerosols, but require knowledge of the proportion of surface area comprised of fatty acids for application.