Subzero Temperatures Research Articles

Desolvation Inability of Solid Hydrates, an Alternative Expression for the Gibbs Free Energy of Solvation, and the Myth of Freeze-Drying.

The term "desolvation inability" is proposed in order to describe the alteration of the original chemical structure of a solute ("decomposition") prior to the solvent's full removal upon the heating of the solvate. This behavior has been sporadically reported; however, it is much more frequent, and it is the basis of various, seemingly unrelated, effects/processes, e.g., the vinegar syndrome of cellulose acetate cinematographic films, in thermal energy storage. An explanation and a criterion/index for the prediction of this behavior are provided based on the comparison of the Gibbs free energies of decomposition and desolvation. A new approach for the expression of the Gibbs free energy of desolvation is proposed by reversing the roles of the solute and solvent and by regarding water as the solute rather than as the solvent, while the solute is treated as a solid solvent. This approach results in lower solvation/desolvation Gibbs free energy values. Based on the above, the experimentally observed thermal behavior of three inorganic hydrates is predicted and explained. Theoretically and experimentally, it is supported that decomposition is possible at sub-zero (°C) temperatures and the regarded simultaneous drying and protection of heat-sensitive substances by freeze-drying, at least in some cases, e.g., for the case of gallic acid, is an unverified myth.

Enhancing Mechanical Performance of High-Density Polyethylene at Different Environmental Conditions with Outstanding Foamability through In-Situ Rubber Nanofibrillation: Exploring the Impact of Interface Modification.

In this study, we utilized in situ nanofibrillation of thermoplastic polyester ether elastomer (TPEE) within a high-density polyethylene (HDPE) matrix to enhance the rheological properties, foamability, and mechanical characteristics of the HDPE nanocomposite at both room and subzero temperatures. Due to the inherent polarity differences between these two components, TPEE is thermodynamically incompatible with the nonpolar HDPE. To address this compatibility issue, we employed a compatibilizer, styrene/ethylene-butylene/styrene copolymer-grafted maleic anhydride (SEBS-g-MA), to reduce the interfacial tension between the two blend components. In the initial step, we prepared a 10% masterbatch of HDPE/TPEE with and without the compatibilizer using a twin-screw extruder. Subsequently, we processed the 10% masterbatch further through spun bonding to create fiber-in-fiber composites. Scanning electron microscopy (SEM) analysis revealed a significant reduction in the spherical size of HDPE/TPEE particles following the inclusion of SEBS-g-MA, as well as a much smaller TPEE nanofiber size (approximately 60-70 nm for 5% TPEE). Moreover, extensional rheological testing revealed a notable enhancement in extensional rheological properties, with strain-hardening behavior being more pronounced in the compatibilized nanofibrillar composites compared to the noncompatibilized ones. SEM images of the foam structures depicted substantial improvement in the foamability of HDPE in terms of the cell size and density following the nanofibrillation process and the use of the compatibilizer. Ultimately, the in situ rubber fibrillation and enhancement of HDPE and TPEE interface using a compatibilizer led to increasing the HDPE ductility at room and subzero temperatures while maintaining its stiffness.

Probing Chemical Equilibrium in Frozen Sodium Phosphate Buffer Solution by 31P Solid-State NMR.

Phosphate buffers are crucial for cryopreservative stability in pharmaceuticals, food processing, biomedical sciences, and biology. However, their freeze concentrates lack quantitative characterization, especially regarding the physicochemical properties of phosphate salt species in equilibrium at subzero temperatures. This study employs 31P solid-state NMR (ssNMR) to analyze frozen sodium phosphate (NaP) solutions, providing insights into phase composition, ionic strength, and pH. For the first time, we have directly quantified phosphate species in frozen NaP buffer, including crystallized disodium phosphate dodecahydrate (Na2HPO4·12H2O) content and the concentrations of H2PO4- and HPO42- in the freeze concentrate. This enabled the calculation of the pH as well as the ionic strength in the freeze concentrate. Trehalose effectively mitigated pH shifts in buffer solutions by preventing the selective crystallization of salt, a spectroscopic phenomenon not previously observed experimentally.

Bench studies of the influence of operating conditions on operational safety of steam turbine air-cooled condensers at subzero outdoor temperatures

Condensation processes are widely spread in various branches of industrial production and, in particular, in electric power industry, where, according to experts' data, heat removal in thermodynamic cycles of steam turbine plants (STP) operating as part of combined cycle plants (CCP) and autonomously, in the 21st century will be exercised mainly in air-cooled condensers (ACC). At the same time, the use of ACC as a part of steam turbine units operating in climatic conditions typical for the Russian Federation (RF) determines the relevance of problems to be solved to ensure their safe operation at subzero outside temperatures. One of these challenges is to ensure long-term safe operation of the ACC, with the formation of ice on the inner surface of the heat exchange pipes minimized. To conduct the research, Steam Turbine Air-Cooled Condenser research and training stand was used. The paper presents the combined circuit and measurement diagram of the research and training stand, and gives a brief description of the design of the ACC. Experimental modes have been determined, and methods for processing experimental data and analyzing the results obtained have been developed. As part of the study, experiments were carried out on 18 steady-state operation modes of the stand. At the same time, the values of the main operational parameters of the ACС varied within the following limits: the temperature of the cooling air at the ACС inlet — from minus 11,5°C to minus 1,8°C; the cooling ratio — from 176 to 790. The relative weight content of air in the vapor varied from 0,051% to 0,12%. An analysis of the obtained results was carried out, on the basis of which a relationship was established that allows, for various negative temperatures of the cooling air at the inlet to the ACС, to determine the highest value of the cooling ratio at which its long-term safe operation is possible. An algorithm for using the obtained relationship is given.

A simple model of the soil freezing characteristic curve for saline soils with two freezing stages

Soil freezing characteristic curve (SFCC) describes the relationship between unfrozen water and subzero temperature, which is of significance for the simulation of heat, water, salt migration in cold regions. There are two phase transition stages in some saline soils, which is often explained by the phase equilibrium of bulk solution. However, the second phase transition and chemical characteristic of solute are ignored in the current coupling numerical models for freezing-thawing soils. The newest SFCC methods considering abovementioned two items are not applicable to numerical models due to complex calculation or irregularly changeable parameters. To solve those problems, a simple model was proposed in this paper. Firstly, the complete phase diagram of pore solution at icing stage was speculated from published experimental SFCCs of saline soils. Then, the temperatures of saline soils at freezing and eutectic points were quantitative expressed with phase diagram of bulk solution and SFCC of nonsaline soil, and the criteria determining whether single icing stage or icing-eutectic stage occurs in saline soils was proposed. A synthetic index was used to describe the effects of soil matrix and initial solute concentration on the exponent. Only three constant parameters were introduced to reflect the soil matrix effect on phase transition points of pore solution and the influence of chemical characteristic of solute on pore water freezing process. Finally, the model was validated by the experimental data of saline soils and showed good results and ease of use compared to a widely used theorical model for saline soil with single freezing stage and other two models both considering two phase transitions. The work provides a deeper view of freezing process in saline soils and promote the improvement of numerical simulation effect for heat, water and salt migration in seasonal freezing-thawing areas.

Reconfiguring the Electrolyte Network Structure with Bio‐Inspired Cryoprotective Additive for Low‐Temperature Aqueous Zinc Batteries

AbstractDespite promising performance at ambient temperature, the development of aqueous zinc batteries is jeopardized by the freeze of aqueous electrolytes and deteriorative electrode‐electrolyte interphase at low temperatures. Herein, inspired by the cryoprotective mechanism of extracellular polysaccharides in biological organisms, a quaternized galactomannan polysaccharide (q‐GPA) is proposed as a cryoprotective additive for improving low‐temperature performance of aqueous zinc batteries. Mechanistic studies revealed that a multi‐hydroxyl galactomannan backbone can substantially attenuate the activity of water molecules through the reconfiguration of the hydrogen bond network, which inhibits ice crystal formation at subzero temperatures and thus depress the freezing point of the electrolyte. Meanwhile, the quaternary ammonium groups tethered on the q‐GPA skeleton are intended to neutralize the interfacial electric field through electrostatic repulsion, thereby accelerating Zn2+ deposition kinetics and prohibiting zinc dendrite growth. Impressively, the q‐GPA–modified electrolyte enables an extended lifespan of over 1700 h in Zn||Zn symmetric battery at a high current density of 3 mA cm−2 and an ultralong cycle life of 5000 cycles with a capacity retention of 99.2% in the Zn||Na2V6O16·1.5H2O (NVO) full battery at −30 °C. This work provides unprecedented possibilities for optimizing the electrolyte formulation of low‐temperature aqueous batteries.

Fabricating supramolecular lyotropic crystals at subzero temperatures: The pivotal role of antifreeze and nanostructures

The classical lyotropic liquid crystals (LLCs) prepared in pure water are not adapted to subzero temperatures. However, the fabrication of cryogenic LLCs is highly significant for gaining profound insights into self-assembly at extremely low temperatures. A proficient solution strategy, namely introducing alcohol antifreeze to water, faces a challenge in balancing frozen resistance and efficient self-assembly. Herein, we address this unmet challenge by selecting four alcohols with comparable structure to fabricate cryogenic LLCs system through the supramolecular self-assembly of a long-chain non-ionic surfactant. The freezing point, surfactant solubility, and self-assembly behavior at subzero temperatures were investigated. The alcohol antifreeze plays a significant role in depressing water freezing point and governing assembled microstructures. Both experimental and computational results revealed that alcohols bearing more hydroxyl groups are efficient to lower water freezing point while remain the capability to induce surfactant self-assembly. They can realize solvophobic balance and strong hydrogen-bond interactions. A simple approach based on Gordon parameter was established to evaluate the suitability as an anti-freezing medium for amphiphile self-assembly. Strikingly, a new LLCs system capable of withstanding the extremely low temperature of −80 °C was developed, which shows interestingly continuous phase transitions as temperature lowers to subzero. Overall, this study provides valuable insights into cryogenic supramolecular self-assembly, and offers a significant foundation and evaluation method for the selection of appropriate alcohol antifreeze for cryogenic self-assembly.

Preservation of Bacillus subtilis' cellular liquid state at deep sub-zero temperatures in perchlorate brines.

Although a low temperature limit for life has not been established, it is thought that there exists a physical limit imposed by the onset of intracellular vitrification, typically occurring at ~-20 °C for unicellular organisms. Here, we show, through differential scanning calorimetry, that molar concentrations of magnesium perchlorate can depress the intracellular vitrification point of Bacillus subtilis cells to temperatures much lower than those previously reported. At 2.5 M Mg(ClO4)2, the peak vitrification temperature was lowered to -83 °C. Our results show that inorganic eutectic salts can in principle maintain liquid water in cells at much lower temperatures than those previously claimed as a lower limit to life, raising the prospects of active biochemical processes in low temperature natural settings. Our results may have implications for the habitability of Mars, where perchlorate salts are pervasive and potentially other terrestrial and extraterrestrial, cryosphere environments.

Hofmeister-Effect-Driven Hybrid Glycerogels for Perfect Wide-Temperature Shape Fixity and Shape Recovery in Soft Robotics Applications.

Shape memory gels have emerged as crucial elements in soft robotics, actuators, and biomedical devices; however, several problems persist, like the trade-off between shape fixity and shape recovery, and the limited temperature range for their application. This article introduces a new class of shape memory hybrid glycerogels (GGs) designed to address these limitations. The well-modulated internal structure of the GGs, facilitated by the Hofmeister salting-out effect, strategically incorporates a higher crystallite content, abundant crosslinking points, and a high elastic modulus. Unlike reported shape memory gels, the GG exhibits a perfect triple-step shape memory behavior in air with 100% shape fixity in a wide programming temperature range (75-135°C) and simultaneously achieves 100% shape recoverability. The gel recovers its shape at -40°C under near-infrared light across a wide programming temperature range (25-135°C), showing unexpected initiation even at subzero temperatures. Inspired by the mechanics of composite structures, a method is proposed to integrate the GG seamlessly with a shape memory alloy, which further expands the temperature range that yields perfect shape memory properties. Finally, two light-controlled fluttering and crawling soft robot prototypes are engineered to illustrate the versatility and potential applications of the composite gel in soft robotics.

The stuff of memories: Planning hindsight in animal cryobanks.

Biobanks are becoming ubiquitous infrastructures in zoology and other non-human life sciences. They promise to store frozen research samples for the long term for future use. That use remains speculative but nevertheless needs to be anticipated. Following the establishment of a physical and digital infrastructure for frozen samples in an animal biobanking project, this article explores how the future is anticipated to remember the past, and how frozen objects are shaped accordingly. Situating the biobank between mundane freezing routines in a research lab and the 'dry' and 'wet' collections of natural history museums, I argue that frozen research objects need to be conserved in two separate ways. The unavailability of cryo-objects in cold storage forces researchers to store materials independently of metadata, while retaining a link between them that allows for their reunion after thawing. The result is a split object, leading a double life at sub-zero and room temperature, linked only through the surface of special plastic containers. Following the making of such split objects, this article offers an elaboration of Radin's 'planned hindsight' as well as a reflection on the universality and particularity of biobanks as standardized scientific memory.

Влияние расстояния между выпускными выработками на потери руды от смерзания при выпуске

The paper considers the results of experimental studies of changes in the ore losses caused by congealing in the stoping zone during the drawing process in conditions of subzero temperatures in underground mining of eore deposits in the cryolithic zone, depending on the distance between the draw holes. Three series of experiments were performed using trench-shaped test benches with various distances betwen the draw holes. The distances between the access crosscuts were assumed to be 180, 200 and 220 mm, which is equal to 9, 10 and 11 m in actual conditions according to the adopted geometric scale of modeling. The results of modeling the bottom drawing in conditions of subzero temperatures, similar to the conditions of the underground ore mining in the cryolithic zone, showed that changes in the distance between the draw holes with the trench-shaped design of the bottom block have a significant impact on the extraction rates. When the distance between the draw holes is increased from 9 m to 11 m, the ore loss due to congealing during the draw phase rises from 24% to 42%. Experimental studies have shown that the increased distance between the draw holes in conditions of subzero temperatures in the bottom area leads to increased ore losses caused by congealing in the stoping area.

The Effect of Hyperbaric Storage on the Nutritional Value and Retention of Certain Bioactive Proteins in Human Milk.

Human milk (HM) contains the essential macronutrients and bioactive compounds necessary for the normal growth and development of newborns. The milk collected by human milk banks is stored frozen and pasteurized, reducing its nutritional and biological value. The purpose of this study was to determine the effect of hyperbaric storage at subzero temperatures (HS-ST) on the macronutrients and bioactive proteins in HM. As control samples, HM was stored at the same temperatures under 0.1 MPa. A Miris HM analyzer was used to determine the macronutrients and the energy value. The lactoferrin (LF), lysozyme (LYZ) and α-lactalbumin (α-LAC) content was checked using high-performance liquid chromatography, and an ELISA test was used to quantify secretory immunoglobulin A (sIgA). The results showed that the macronutrient content did not change significantly after 90 days of storage at 60 MPa/-5 °C, 78 MPa/-7 °C, 111 MPa/-10 °C or 130 MPa/-12 °C. Retention higher than 90% of LYZ, α-LAC, LF and sIgA was observed in the HM stored at conditions of up to 111 MPa/-10 °C. However, at 130 MPa/-12 °C, there was a reduction in LYZ and LF, by 39 and 89%, respectively. The storage of HM at subzero temperatures at 0.1 MPa did not affect the content of carbohydrates or crude and true protein. For fat and the energy value, significant decreases were observed at -5 °C after 90 days of storage.

Evolution of pore structure and analysis of freeze damage in granite during cyclic freeze-thaw using NMR technique

Understanding the changes in a rock's pore structure and unfrozen water content during the freeze-thaw (F-T) process is crucial for unraveling the F-T damage mechanism of rock masses. This study employs nuclear magnetic resonance (NMR) tests to resolve the pore structure characteristics of granite specimens subjected to varying numbers of F-T cycles (0, 50, 100, and 200 cycles). Throughout the F-T process, unfrozen water (comprising bound water and free water) is quantified through T2 distribution curves, and the freeze-induced strain at different subzero temperatures is investigated. The results reveal that F-T cycles lead to the expansion of pore sizes from small to large, reducing the complexity of the pore structure. Moreover, an increased proportion of meso- and macro-pores accelerates the rate of water-ice phase transition in granite specimens, resulting in intensified freeze damage and greater maximum freeze-induced strain. Additionally, when previously damaged granite is refrozen, the presence of free water induces higher levels of freeze-induced strain. This research comprehensively analyzes the F-T damage process in granite, laying a crucial theoretical foundation for understanding freeze damage mechanisms in rock engineering.

Probing the interaction between biomolecules under sub-zero temperature conditions by electrophoresis in ice grain boundaries

BackgroundPsychrophiles can survive under cryogenic conditions because of various biomolecules. These molecules interact with cells, ice crystals, and lipid bilayers to enhance their functionality. Previous studies typically measured these interactions by thawing frozen samples and conducting biological assays at room temperature; however, studying these interactions under cryogenic conditions is crucial. This is because these biomolecules can function at lower temperatures. Therefore, a platform for measuring chemical interactions under sub-zero temperature conditions must be established. ResultsThe chemical interactions between biomolecules under sub-zero temperature conditions were evaluated within ice grain boundaries with a channel-like structure, which circumvents the need for thawing. An aqueous solution of sucrose was frozen within a microfluidic channel, facilitating the formation of freeze-concentrated solutions (FCSs) that functioned as size-tunable electrophoretic fields. Avidin proteins or single-stranded DNA (ssDNA) were introduced into the FCS in advance. Probe micro/nanospheres whose surfaces were modified with molecules complementary to the target analytes were introduced into the FCS. If the targets have functionalities under sub-zero temperature conditions, they interact with complementary molecules. The chemical interactions between the target molecules and nanospheres led to the aggregation of the particles. The size tunability of the diameter of the FCS channels enabled the recognition of aggregation levels, which is indicative of interaction reactivity. The avidin–biotin interaction and ssDNA hybridization served as models for chemical interactions, demonstrating interactivity under sub-zero temperature conditions. The results presented herein suggest the potential for in situ measurement of biochemical assays in the frozen state, elucidating the functionality of bio-related macromolecules at or slightly below 0 °C. SignificanceThis is the first methodology to evaluate chemical interactions under sub-zero temperature conditions without employing the freeze-and-thaw process. This method has the advantage of revealing the chemical interactions only at low temperatures. Therefore, it can be used to screen and evaluate the functionality of cryo-related biomolecules, including cold-shock and antifreeze proteins.

High-performance and frost-resistance MXene co-ionic liquid conductive hydrogel printed by electrohydrodynamic for flexible strain sensor

Conductive hydrogels with high performance and frost resistance are essential for flexible electronics, electronic skin, and soft robots. Nonetheless, the preparation of hydrogel-based flexible strain sensors with rapid response, wide strain detection range, and high sensitivity remains a considerable challenge. Furthermore, the inevitable freezing and evaporation of water in sub-zero temperatures and dry environments lead to the loss of flexibility and conductivity in hydrogels, which seriously limits their practical application. In this work, ionic liquids (ILs) and MXene are introduced into gelatin/polyacrylamide (PAM) precursor solution, and a PAM/gelatin/ILs/MXene/glycerol (PGIMG) hydrogel-based flexible strain sensor with MXene co-ILs ion–electron composite conductive network is prepared by combining the electrohydrodynamic (EHD) printing method and in-situ photopolymerization. The introduction of ILs provides an ionic conductive channel for the hydrogel. The introduction of MXene nanosheets forms an interpenetrating network with gelatin and PAM, which not only provides a conductive channel, but also improves the mechanical and sensing properties of the hydrogel-based flexible strain sensor. The prepared PGIMG hydrogel with the MXene co-ILs ion–electron composite conductive network demonstrates a tensile strength of 0.21 MPa at 602.82 % strain, the conductivity of 1.636 × 10−3 S/cm, high sensitivity (Gauge Factor, GF = 4.17), a wide strain detection range (1–600 %), and the response/recovery times (73 ms and 74 ms). In addition, glycerol endows the hydrogel with excellent freezing (−60 °C) and water retention properties. The application of the hydrogel-based flexible strain sensor in the field of human motion detection and information transmission shows the great potential of wearable devices, electronic skin, and information encryption transmission.

Numerical studies of pull-through failures in cold-formed steel battens at sub-zero temperatures

Cold-formed steel (CFS) roof and wall cladding systems comprising thin, high strength steel battens and claddings are popularly used in the building industry worldwide. These cladding systems have been observed to encounter failures under combined snow and strong wind actions during blizzards. Significant damage is caused by the disengagement of the entire roof/wall cladding when localised pull-through failures occur in thin-walled steel battens at the batten to truss/rafter screw connections due to a splitting mechanism around the screw head. This study uses detailed finite element analyses of the pull-through failures of a range of batten configurations under combined wind and sub-zero temperature conditions. Suitable finite element models capable of simulating the pull-through failures of battens were developed and validated using experimental results, followed by detailed numerical parametric studies. The main outcomes of this study are accurate design equations and capacity reduction factors essential for predicting the pull-through capacities of CFS battens at sub-zero temperatures.

Mechanical properties and damage development in glass-fiber epoxy laminates subjected to tensile loading at sub-zero temperatures

In various structural applications polymer composites are exposed to sub-zero and even cryogenic temperatures which may initiate of microstructural damage. To anticipate these events, one needs to understand the behavior of composites in a sub-zero environment. This study focuses on damage initiation and accumulation, and its influence on the properties of cross-ply glass fibers epoxy composites at sub-zero temperatures. The effect of bromine modification of epoxy, and the dissolution in an organic solvent on the mechanical performance of the produced composite is also investigated. To evaluate the influence of a sub-zero environment on the mechanical performance of glass fiber epoxy laminates, tensile tests in a sub-zero environment of unconditioned specimens were carried out. The quasi-static tensile tests were performed to measure the elastic modulus of the composites while loading-unloading experiments were performed to monitor the initiation (and accumulation) of microstructural damage and its influence on the stiffness of glass fiber epoxy laminates. The results of cryogenic damage and fracture in the laminates are discussed with a focus on the degradation of properties of glass fiber crucial for their use in structural applications: strength and stiffness.

Low-temperature characterization of inkjet printed silver nanoparticles on polyimide substrate for the temperature range of −5°C to −75°C

Drop-on-demand inkjet printing is an additive manufacturing method capable of fabricating electronic devices using custom geometries at a relatively low cost. Drop-on-demand using silver nanoparticles has been extensively studied to produce devices and sensors that operate at room conditions. This work investigates the survivability and performance of inkjet-fabricated silver traces tested in an electric circuit at a subzero temperature environment from −5℃ to −75℃. Results show that printed Ag nanoparticles exhibit a similar thermal resistance coefficient compared to literature values of bulk nanostructured Silver at subzero temperatures, rendering this additive manufacturing technology a strong candidate for creating microelectronic devices for low-temperature aerospace applications.

Bond behavior of CFRP-strengthened steel structures and its environmental influence factors: a critical review

Adhesively bonded carbon fiber-reinforced polymer (CFRP) systems have shown great promise for strengthening damaged steel structures, offering potential enhancements in the longevity of steel structures. However, the degradation of bond performance of CFRP under harsh environments remains a critical concern for researchers and engineers, as it may significantly impact the efficiency and durability of bonded CFRP strengthening systems. This paper presents a comprehensive review of the impact of key characteristics of bonded joints, such as the CFRP modulus, adhesive performance, bond length, adhesive layer thickness, and bond joint geometry, on the bonding performance of CFRP strengthening systems. Additionally, the influences of environmental factors, including elevated and sub-zero temperatures, moisture, corrosion, humidity, wet-dry cycles, ultraviolet radiation, and freeze-thaw cycles on the bond behavior, were also reviewed. By synthesizing and analyzing existing research insights into the effects of reinforcement materials, bond joint design parameters, and environmental factors on bonding performance, this review article attempts to enhance practitioners' understanding of bond behavior in this field and also provides guidance for future research.

Ice-Nucleating Gut Microbes in Insects: A Scoping Review

(1) Background. At subzero temperatures, water crystallizes on ice nucleation agents. Researchers have identified ice-nucleating microbes (INMs) in insect digestive tracts that can raise the insect’s supercooling point, causing freezing at higher temperatures but slower rates. For freeze-tolerant insects, such gut microbes should allow for slower freezing away from tissues and higher survival rates. For freeze-susceptible insects, however, such microbes could cause a fatal freeze at higher temperatures, and could possibly be used as biocontrol. (2) Methods. A first-ever scoping review was carried out of research on insect-associated INMs, from observational studies attempting to isolate these microbes, to experimental studies applying them and checking for increased mortality. (3) Results. Relatively few research groups have studied insect-associated INMs in any capacity. (4) Conclusions. Several authors hypothesized that such microbes are probably abundant, and their contribution to ice nucleation activity in insects is under-reported. Biocontrol assays using ice-nucleating microbes showed promise, but a risk to non-target organisms has been experimentally confirmed. Future surveys of insect–microbe interactions using molecular tools are likely to reveal new examples, if not new microbe species capable of ice nucleation.