Dissemination of IT for the Promotion of Materials Science (DoITPoMS)

# Avoidance of crystallization by freeze resistance

Many organisms exist in habitats where the temperatures fall below the freezing point of water. As previously explained, the formation of ice crystals in cells is lethal and many species have therefore evolved to prevent ice crystals forming in cells.

There are two different types of resistance to freezing temperatures: freeze avoidance and freeze toleration.

### Freeze avoidance

During freezing of water ice crystals nucleate and grow. For pure water, homogenous nucleation occurs at ~ 40 K beneath the thermodynamic freezing point. This is a substantial supercooling, but in most cases, freezing occurs above –40°C due to heterogeneous nucleation.

One way to avoid freezing is to discourage heterogeneous nucleation. Some frost-hardened woods achieve this by dispersing the water in cells, and by the lack of nucleation on the cell walls. As a result, the water in their cells does freeze at –40°C.

Fish, insects and some plants that live in arctic regions have evolved to produce antifreeze proteins, which inhibit the growth of ice crystals by adsorption to the ice surface. Adsorption of these antifreeze proteins prevents crystal growth on the primary growth directions, forcing growth to occur parallel to the secondary axes. This inhibits the formation of stable ice crystals and lowers the kinetic freezing temperature.

### Freeze toleration

An alternative to freeze avoidance is to promote the freezing of extracellular liquid. This protects the cells in two ways:

• By the release of latent heat into the cells, which prevents their temperature from decreasing.
• By drawing water out of the cells and decreasing the temperature at which ice forms. This depression of the liquidus line at higher sucrose concentrations can be seen in the sucrose-water phase diagram.
• Ultimately dehydration of the cells leads to glass formation.

Many biological systems promote heterogeneous nucleation by the presence of a variety of Ice Nucleating Agents (INAs). These INAs may either be adaptive or incidental. Adaptive INAs, which are discussed below, are present in order to promote heterogeneous nucleation, whereas incidental INAs (for example, features such as cell walls) promote heterogeneous nucleation only as a normally unwanted side effect.

Some organisms have evolved to produce adaptive INAs, which nucleate ice crystals between the cells. These INAs can reduce the nucleation supercooling to as little as 1°C. Examples of these are the giant rosette plant (lobelia telekii) which grows on Mount Kenya, and the northern wood frog (rana sylvatica) which lives in Canadian forests. The northern wood frog's body contains 35-45% ice during the winter months.

Adaptive INAs are generally large proteins with molecular weights of up to 30,000 atomic mass units. The amino acids within the proteins are ordered, forming a template for ice. Thus a thin layer of ice can always form on the surface of an INA. However, this will not lead to spontaneous ice growth unless the INA is of a certain critical size. If the INA is assumed to have a circular surface with radius R, then free ice growth will occur only when R is greater than or equal to r*. The critical radius, r*, is given by the equation:

$r* = - {{2\gamma } \over {\Delta {S_v}\Delta T}}$

where γ is the interfacial energy per unit area, and ΔSvΔT is the free energy of solidification per unit volume. Nucleation will occur when the supercooling ΔT satisfies the condition:

$\Delta T \ge - {{2\gamma } \over {\Delta {S_v}R}}$

A larger INA (greater R) therefore gives a smaller required supercooling and a higher nucleation temperature.