Bacterial Ice Nucleation in Monodisperse D₂O and H₂O-in-Oil Emulsions

Abstract

Ice nucleation is of fundamental significance in many areas, including atmospheric science, food technology, and cryobiology. In this study, we investigated the ice-nucleation characteristics of picoliter-sized drops consisting of different D2O and H2O mixtures with and without the ice-nucleating bacteria Pseudomonas syringae. We also studied the effects of commonly used cryoprotectants such as ethylene glycol, propylene glycol, and trehalose on the nucleation characteristics of D2O and H2O mixtures. The results show that the median freezing temperature of the suspension containing 1 mg/mL of a lyophilized preparation of P. syringae is as high as −4.6 °C for 100% D2O, compared to −8.9 °C for 100% H2O. As the D2O concentration increases every 25% (v/v), the profile of the ice-nucleation kinetics of D2O + H2O mixtures containing 1 mg/mL Snomax shifts by about 1 °C, suggesting an ideal mixing behavior of D2O and H2O. Furthermore, all of the cryoprotectants investigated in this study are found to depress the freezing phenomenon. Both the homogeneous and heterogeneous freezing temperatures of these aqueous solutions depend on the water activity and are independent of the nature of the solute. These findings enrich our fundamental knowledge of D2O-related ice nucleation and suggest that the combination of D2O and ice-nucleating agents could be a potential self-ice-nucleating formulation. The implications of self-nucleation include a higher, precisely controlled ice seeding temperature for slow freezing that would significantly improve the viability of many ice-assisted cryopreservation protocols.

Controlled ice nucleation using freeze-dried Pseudomonas syringae encapsulated in alginate beads

“…Nevertheless, the current ice-nucleating methods suffer from a number of disadvantages such as the lack of consistency and standardization in the case of manual operations [16], the cumbersome process for a large number of samples [24], and the necessity of electric and/or magnetic fields. To overcome some of these limitations, ice-nucleating agents (INAs) have been extensively explored, such as mineral dust kaolinite [15, 17], silver iodide [3, 23], and bacterium Pseudomonas syringae [19, 24].…”

“…To overcome some of these limitations, ice-nucleating agents (INAs) have been extensively explored, such as mineral dust kaolinite [15, 17], silver iodide [3, 23], and bacterium Pseudomonas syringae [19, 24]. It is generally suggested that INAs induce ice nucleation heterogeneously because the microscopic structure of the INA particle surface resembles the ice crystalline structure [24]. For example, silver iodide particles (typically 1 μm in diameter) have the same unit cell dimension as ice crystals, being able to induce the freezing of pure water at a temperature up to −4°C [3, 23].…”

“…The best characterized of these is Pseudomonas syringae , a widely distributed bacterial epiphyte of plants [1]. Our previous study has systematically investigated the ice nucleation kinetics of picoliter-sized drops of heavy water (D 2 O) and light water (H 2 O) mixtures with freeze-dried P. syringae [24]. The results showed that the median freezing temperature of the 1 mg/ml P. syringae suspension prepared in 100% D 2 O was as high as −4.6°C [24].…”

“…Our previous study has systematically investigated the ice nucleation kinetics of picoliter-sized drops of heavy water (D 2 O) and light water (H 2 O) mixtures with freeze-dried P. syringae [24]. The results showed that the median freezing temperature of the 1 mg/ml P. syringae suspension prepared in 100% D 2 O was as high as −4.6°C [24]. A recent interface-specific sum frequency generation (SFG) spectroscopy and molecular dynamics simulation study revealed that the hydrogen bonds formed at the water-bacterium interface imposed structural ordering on the adjacent water network facilitating ice nucleation [19].…”

“…One of the major advantages of INAs is their self-ice-seeding capability, that is heterogeneously initiating ice formation at their characteristic temperatures without manual or instrumental interruptions [24]. But the use of INAs in biological systems may introduce concerns about their biocompatibility, degradability, toxicity, recovery and ease of use.…”