On the bioprotective effects of 3-hydroxybutyrate: Thermodynamic study of binary 3HB-water systems

Abstract

Microorganisms must face various inconvenient conditions; therefore, they developed several approaches for protection. Such a strategy also involves the accumulation of compatible solutes, also called osmolytes. It has been proved that the monomer unit 3-hydroxybutyrate (3HB), which is present in sufficient concentration in poly(3-hydroxybutyrate) (PHB)-accumulating cells, serves as a chemical chaperone protecting enzymes against heat and oxidative stress and as a cryoprotectant for enzymes, bacterial cells, and yeast. The stress robustness of the cells is also strongly dependent on the behavior and state of intracellular water, especially during stress exposure. For a better understanding of the protective mechanism and effect of strongly hydrophilic 3HB in solutions at a wide range of temperatures, a binary phase diagram of system sodium 3HB (Na3HB)-water in equilibrium and the state diagrams showing the glass transitions in the system were constructed. To investigate the activity of water in various compositions of the Na3HB/water system, three experimental techniques have been used (dynamic water sorption analysis, water activity measurements, and sorption calorimetry). First, Na3HB proved its hydrophilic nature, which is very comparable with known compatible solutes (trehalose). Results of differential scanning calorimetry demonstrated that Na3HB is also highly effective in depressing the freezing point and generating a large amount of nonfrozen water (1.35 g of water per gram of Na3HB). Therefore, Na3HB represents a very effective cryoprotectant that can be widely used for numerous applications.

Significance

The comprehensive study of the phase behavior of sodium 3-hydroxybutyrate (Na3HB) in aqueous mixtures has shed new light on the recently revealed bioprotective role of this compound in poly(3-hydroxybutyrate)-accumulating microorganisms. The outstandingly hydrophilic nature of 3HB is shown, which is comparable with, but in some perspectives (solubility, water activity decreased in solution) even better than some well-recognized compatible solutes such as trehalose. This is of crucial importance also from the view of its potential application in the technological fields in which stabilization of biological molecules is required (e.g., cryopreservation, food preservation, cosmetics). The study also revealed that 3HB can form, depending on the conditions, at least two different crystalline forms: anhydrous crystal and crystalline dihydrates.

Introduction

Natural cryoprotectants (or cryoprotective agents (CPAs)) have been known since the nineteenth century, when the earliest concepts of cryoprotection were established. Nowadays, cryobiology is an essential field with high application in biotechnology and modern medicine, where macromolecules, cell components, and cells are protected against freezing and/or desiccation by cryoprotective additives. The presence of CPAs stabilizes the activity of enzymes and promotes a glassy state during freezing, which is crucial for the viability of cells. CPAs are generally classified as 1) penetrating CPAs (capable of diffusing through the plasma membrane), revealing common properties such as low molecular weight, high solubility, and nontoxicity, and 2) non-penetrating CPAs, which are generally long-chain water-soluble polymers with high osmotic coefficients (1,2). Many of the penetrating CPA (e.g., trehalose, ectoine, betaine) combine the cryoprotection with shielding of labile cellular components against other environmental stress factors, including high temperature (3), oxidative damage (4), and/or extreme pH (5). The mechanism of the universal protective performance of these compounds—often referred to as compatible solutes or chemical chaperones—is not clearly understood; nevertheless, it is believed that they can stabilize labile biopolymers (e.g., proteins) by affecting their hydration in the cell (1,6). Therefore, understanding how a biomolecule affects the activity of water in aqueous solutions seems to represent an essential step to evaluating or understanding its bioprotective potential.

Microorganisms employ diverse approaches to protect themselves when exposed to adverse environmental conditions. One of the common protective mechanisms, which numerous strains of bacteria and even some Archaea utilize, is the capability of production and accumulation of poly(3-hydroxybutyrate) (PHB) (7). The role of intracellular PHB had long been attributed only to the storage of energy and carbon; however, it has recently been revealed that intracellular PHB granules protect microbial cells against various stressors, such as high temperature, osmotic shock, UV exposure, and oxidative pressure (8,9,10). The principle of this complex protection seems to be based both on PHB metabolism and on the unique structural and physicochemical properties of the PHB granules (7). Furthermore, PHB-accumulating microorganisms possess a high intracellular pool of 3-hydroxybutyrate (3HB) monomers (11) that also play their specific protective role. It was recently found that 3HB serves as a chemical chaperone protecting enzymes against overheating and oxidative stress (12), a compatible solute protecting bacteria against the high salinity of the environment (13) and also a cryoprotectant for enzymes (lipase), yeasts (Saccharomyces cerevisiae), and bacterial cells (Cupriavidus necator) (14).

It is very unlikely that the stabilizing effect of 3HB for biological samples under such fundamentally distinct stress conditions as cold and high temperatures, or in an oxidative environment, respectively, could be attributed to a single physical or chemical mechanism of protection. On the other hand, it is indisputable that the state of intracellular water plays an essential role under all of these circumstances. Protein stabilization by compatible solutes has most often been ascribed to the so-called preferential hydration phenomenon (1), which combines a general colligative decrease of water activity in a solution with a solute-specific preferential binding or exclusion of the solute molecules from the immediate surface of the protein. The role of water activity is even more obvious whenever exposure to harmful conditions results in cell desiccation. This is not only provided by a high osmolality of the environment but also represents a harmful consequence of cell freezing under a slow cooling rate, where the formation of extracellular ice concentrates the solutes outside the cells, which results in “freeze-dehydration” (15). On the other hand, cell freezing under higher cooling rates most often results in cell injury via intracellular ice formation, once again strongly dependent on the thermodynamic state of the cellular water (16). Finally, a decrease in water availability is known to be accompanied by disruption of electron transfer systems and an increased generation of reactive oxygen species resulting in oxidative stress manifestations such as metabolic enzyme inactivation and damage to membrane lipids and/or nucleic acids (17).

Hence, in the present work, we aim at investigating the interactions of sodium 3HB (Na3HB), with water, especially at low and subzero temperatures. Understanding of behavior of Na3HB in water solutions is not only essential for revealing its cryoprotective potential but it might also contribute to an understanding of its general protective action for various biomolecules and cells. Two distinct experimental strategies were used: first, hydration of Na3HB was studied using a combination of three different methods of sorption analysis, and, second, phase transitions in aqueous solutions of Na3HB were studied via differential scanning calorimetry (DSC) under equilibrium and at non-equilibrium conditions, respectively.

Materials and methods

Preparation of samples

Na3HB was obtained by Sigma-Aldrich with purity ≥99.0%, CAS number 150-83-4. Before all hydration experiments, Na3HB was dried in a vacuum in contact with 3-Å molecular sieves for at least 24 h at room temperature. Samples of various ratios of Na3HB and water were obtained either by direct pipetting of water to dried Na3HB or hydrated at a controlled relative humidity (RH) in desiccators with saturated salt solutions from several minutes to hours at 25°C. The following salts were used: K₂SO₄ (97.3% RH), KCl (84.34% RH), NaCl (75.29% RH), Mg(NO₃)₂ (52.89% RH), and MgCl₂ (32.78% RH) (18).

Determination of water activity

Dynamic vapor sorption

For vapor sorption analysis, dynamic vapor sorption (DVS) Q5000 SA (TA Instruments, United States) was used. Initially, the dried Na3HB sample was conditioned for 60 min at 60°C at 0% RH and then the % RH value was increased in a stepwise manner covering the range from 40% RH to 70% RH with a 5% RH step. At each % RH level, the sample was kept for 5 h and its weight was recorded continuously. Only the data from % RH steps where the sample weight was equilibrated at the constant value were used for further evaluation. The DVS experiment was repeated for various temperatures (5°C–40°C). To determine the critical level of % RH, where the dry Na3HB begins to absorb the hydration water, the stepwise % RH tests described above was supplemented by the ramp test where the % RH level was increased linearly with the 0.1 % RH/min rate.

Static activity measurements with commercial water activity analyzer

The activity of water in the hydrated Na3HB samples with various Na3HB/water contents was determined at 25°C by commercial resistive electrolytic analyzer LabMaster-a_w (Novasina, Switzerland). Before the analysis of Na3HB/water samples, the analyzer was calibrated by commercial reusable SAL-T calibration standards.

Sorption calorimetry

The sorption calorimetry experiment was performed at 25°C in the double twin calorimeter according to (19), where the dried sample of Na3HB was loaded into the upper chamber (called sorption chamber) and the lower chamber was filled with pure water. This method provides the values of water activity and the corresponding equilibrium water content in the sorbent (Na3HB) as calculated according to (20) from the thermal powers determined separately in both chambers of the twin calorimeter.

DSC

Experiments with Na3HB containing different amounts of water were performed by DSC (DSC 1 Mettler Toledo, Switzerland). For calibration of heat flow and temperature, indium in a hermetically sealed aluminum pan (melting point: 156.6°C; ΔH = 28.45 J/g) and an empty sealed aluminum pan as a reference were used. As the purge gas, dry nitrogen with a gas flow of 80 mL/min was used.

First, samples were loaded into an aluminum pan, hermetically sealed, and cooled down (10°C/min) to a minimal temperature, selected according to the phase transitions to be investigated. Based on the previous optimization of the DSC procedure for Na3HB/water samples, the lowest temperature of −70°C was set in the measurements used for the construction of the phase diagram in equilibrium to avoid the glass transition of the sample. In contrast, the minimal temperature of −90°C was used wherever the glass transitions of the Na3HB/water systems were primarily investigated. After the isothermal conditioning step (5 min), the sample was then heated up to 100°C (first heating scan), cooled down (10°C/min) back to the same minimal temperature, isothermally conditioned (5 min), and, finally, heated up to 100°C again (second heating scan). The heating scans usually ran with a heating rate of 10°C/min; however, lower scan rates (1°C/min, 2°C/min, and 5°C/min) were used wherever the extrapolation to zero heating rate was needed for a proper determination of the endset points of the melting transition (constructing the freezing and solubility curves in Fig. 3). Furthermore, to investigate the equilibrium phase transitions in systems with the Na3HB content ranging from 42 to 65 wt %, scan rate was decreased to the lowest limit of the device (0.2°C/min), and the equilibration time of the isothermal step was 30 min. For the construction of diagrams shown in Figs. 3 and 6, data obtained from the second heating scans were used, except for the measurements with a scan rate of 0.2°C/min, where only a single heating scan was performed.

Figure 3.

The phase diagram in the equilibrium of Na3HB/water complex using DSC (×); sorption calorimetry () and DVS (). All lines are drawn as a guide for the eye to follow the respective phase boundaries.

Figure 6.

State diagram showing glass transitions in Na3HB/water mixtures as determined by DSC. All lines are drawn as a guide for the eye to follow the respective state boundaries. To see this figure in color, go online.

Results and discussion

Water sorption on 3HB

Sorption analysis was performed to evaluate the hygroscopicity of Na3HB at different hydration levels. Sorption isotherms at 25°C (water activity as a function of water content in binary Na3HB/water systems), determined by three independent analytical techniques, are shown in Fig. 1 a. Dynamic water sorption analysis (DVS) is a gravimetry technique based on determining the sample weight after its equilibration at a specific level of RH of the controlled measuring atmosphere. Hereby, the corresponding equilibrium water contents in the sample are determined for different values of water activity (equivalent to the RH). Furthermore, Na3HB hydration at 25°C was analyzed by DVS not only in the step mode but also under a continual increase of RH. The continual mode provides a better resolution of the critical RH at which the hydration of the sample is initiated (Fig. S1, supporting material). The next applied experimental technique (electric hygrometer, LabMaster-aw) is based on an analytical principle opposite to that of DVS; i.e., the values of water activity are determined for samples prepared with controlled water contents. In addition, the sorption microcalorimetry technique, whose instrumentation and methodology are described in detail elsewhere (20), enables simultaneous measurement of both the water activity (RH) and the sample moisture content. Furthermore, as a modified calorimetric method, it also provides differential sorption enthalpy as an additional valuable parameter characterizing the water sorption process.

Figure 1.

(a) Sorption isotherms for water vapor sorption on Na3HB at 25°C determined by Novasina LabMaster (black), DVS (blue), and sorption calorimetry (red) respectively. (b) Enthalpy of hydration of Na3HB as a function of water content as measured by sorption calorimetry at 25°C. (c) Comparison of water vapor sorption isotherms measured by DVS at 25°C (blue) and 40°C (black), respectively.

The results of all three methods are in good agreement, indicating good reproducibility of the water sorption process, and they also provide several interesting findings regarding phase behavior in the Na3HB/water system. Primarily, the stepwise shape of the isotherms shown in Fig. 1 a indicates a complex phase behavior at 25°C with several transitions occurring at specific levels of Na3HB hydration. Starting from the lowest water content, dry crystalline Na3HB does not uptake any water until the relative humidity exceeds a critical level of about 48.5% RH (as determined by DVS in continual mode; see Fig. S1 c, d; supporting material). The low water absorbency of the crystalline forms of cryoprotectants is neither unexpected nor unique. For instance, while the amount of water absorbed by the amorphous form of trehalose (routinely used cryoprotective agent) increases almost linearly with the RH of the atmosphere (21), the crystalline form of trehalose starts to absorb a significant amount of water only at RH above 95%. (22). When exceeding the required limit of water activity, the amount of water absorbed by Na3HB increases to 22.5 wt % (region I in Fig. 1 a). This hydration step is exothermic, as revealed by the results of sorption calorimetry (Fig. 1 b). The threshold amount of water (22.5 wt %) corresponds to two water molecules per one Na3HB. Therefore, the plateau region below this amount of water (depicted as I in Fig. 1 a) represents the equilibrium mixtures of anhydrous crystals and Na3HB dihydrates.

Another region of constant water activity (region II in Fig. 1 a) represents the coexistence of solid Na3HB dihydrate and its saturated solution. From the border concentration of this area (about 45.2 wt % of water), the solubility of Na3HB dihydrate can be determined (54.8 wt % of Na3HB, equivalent to about 120 g of Na3HB per 100 g of water). The solubility is comparable with those of α, α-trehalose (109.6 g per 100 g of water at 30°C (22)). The final step of the sorption isotherm (region III, above 60% RH) represents water uptake by Na3HB solution. In this region, water activity increases with the water content in the solution, whereby the activity coefficient (calculated as a ratio of water activity and molar fraction of water in solution) increases from 0.7 to 0.9. The value of the activity coefficient lower than 1 confirms the hydrophilic nature of Na3HB in its aqueous solution. Moreover, the determined water activities are significantly lower than those published for aqueous solutions of other compatible solutes (e.g., trehalose or sucrose) (23).

To check the effect of temperature on the vapor sorption on Na3HB, DVS analysis was repeated at 40°C. It can be seen in Fig. 1 c that the sorption isotherm shows some similar features but also significant differences compared with that determined at 25°C. Again, a negligible water uptake on dry crystalline Na3HB was observed below 50 % RH (this phenomenon proved to be general as far as we observed it also at temperatures below 25°C, data not shown). Furthermore, also the vapor sorption by Na3HB solution does not significantly differ, showing only slightly lower hydrophilicity of Na3HB (represented by moderately higher activity coefficient of water). On the other hand, only one step change is found in the isotherm, suggesting that no transition between different crystal modifications occurs at the higher temperature. Finally, the solubility of crystalline Na3HB at 40°C increases to 67.1 wt % of 3NaHB (204 g of Na3HB per 100 g of water), which is again higher compared with trehalose (148 g per 100 g of water (22)).

To sum up the results of this experimental part, vapor sorption analyses proved the hydrophilic nature of Na3HB. Quantitative parameters that characterize this hydrophilicity (solubility, water activity decrease in solution) are comparable with those of some well-recognized compatible solutes such as trehalose. Apart from this, the study also revealed that Na3HB can form, depending on the conditions (temperature, RH), at least two different crystalline forms: anhydrous crystal and crystalline dihydrates.

Phase diagram of Na3HB/water at equilibrium

The vapor sorption analysis describes the thermodynamics of Na3HB/water systems at temperatures close to physiological optima. Nevertheless, when discussing the actual bioprotective effects (in particular the cryoprotective ones) it is necessary to analyze the thermodynamics of Na3HB/water binary systems over a much wider range of temperatures. For this purpose, we performed a DSC analysis of the phase transitions occurring during controlled freezing and thawing of various Na3HB/water mixtures. Examples of recorded DSC thermograms and the illustration of their evaluation are shown in Fig. 2. As the result of this evaluation, onset, peak, and endset temperatures were obtained as well as the integral heat of the transition (see the example of ice-melting peak evaluation in Fig. 2 a). For samples with a concentration of Na3HB ranging from 6.3 to 37.8 wt %, two separate endotherm peaks were observed in the thermograms below 0°C upon heating (Fig. 2 b). In this case, the first endothermic peaks belong to the eutectic line where the crystalline hydrates of Na3HB melt, while the second peaks correspond to the melting of ice. On the other hand, thermograms of the samples with a high content of Na3HB (from 72.4 to 81.6 wt %; Fig. 2 c) are featureless until about 30°C, where they show a dominant peak corresponding to melting of solid Na3HB hydrates and a minor peak shifted to a higher temperature attributed to melting of anhydrous Na3HB (marked with the red arrow in Fig. 2 d).

Figure 2.

DSC thermograms of mixtures of Na3HB and water used for construction of equilibrium phase diagram (scanning rates: 10°C/min, scale bars for heat flow are shown). (a) Example of the evaluation of ice melting (Na3HB concentration 30.3 wt %); (b) endotherms at about −27.5°C representing the eutectic points and second endotherms that correspond to ice melting (Na3HB concentrations from 11.3 to 40.5 wt %); (c) endotherms representing the melting of dihydrates and melting of crystalline Na3HB (Na3HB concentrations from 72.4 to 81.6 wt %); (d) magnified endotherms of crystalline Na3HB melting, marked with the red arrow. To see this figure in color, go online.

To provide a complete overview of the equilibrium phase behavior of binary Na3HB/water systems, the results of DSC and the vapor sorption analysis were combined to construct the phase diagram, shown in Fig. 3. To construct the eutectic and peritectic lines, respectively, onset temperatures of the corresponding endothermic peaks were used, while, for the solubility and the ice-melting curves, endset temperatures were utilized. To minimalize the effects of the scan rate on the determined values of the ice melting point and solubility, respectively, DSC measurements were performed at multiple scan rates (see Fig. S2, supporting material) and the endset temperatures were extrapolated to zero scan rate. From the methodological point of view, it was also quite tricky to get any melting data for samples with the content of Na3HB ranging from 42 to 65 wt % because the peak corresponding to the eutectic line was hardly detectable for these samples. The reason is that samples of these compositions showed a high tendency to adopt the glassy state, preventing the sample from crystallizing. Nevertheless, these experimental difficulties have been overcome by equilibrating the sample for half an hour to be above the glass transition point (supporting sample crystallization) and by decreasing the scan rate to the limit of the device (0.2°C/min).

In the equilibrium phase diagram shown in Fig. 3, the water freezing curve represents the most important phase boundary as far as a cryoprotective effect of Na3HB is concerned. The curve ends in the eutectic point that occurs at Na3HB content of 40.5 wt % and temperature of −28.1°C. This means that no ice is formed in solutions above this Na3HB content and temperature higher than −28.1°C. Furthermore, it also indicates the lowest temperature to which the water freezing point can be depressed by the presence of Na3HB. Similar complete or partial phase diagrams have already been published for some compatible solutes and routinely used cryoprotectants such as trehalose (24,25), sucrose (26), or glycerol (27). Compared with these compounds, the effect of Na3HB on the freezing point depression is again quantitatively similar. For instance, published values of eutectic points of trehalose range from −2.5°C to −18.8°C (28,29,30) and for sucrose from −8.5°C to −13.95°C (31). Glycerol can decrease the water freezing point down to −45°C (eutectic point); nevertheless, significantly higher glycerol content is needed (about 65 wt % (28)), while a similar weight content of glycerol (40 wt %) decreases water freezing less than Na3HB (only to −15.4°C (28)).

Based on the DSC data, it was also further confirmed that Na3HB in the solid state does form crystalline dihydrates. For this purpose, the enthalpies of the melting endothermic peak that corresponds to the peritectic line in the phase diagram in Fig. 3 (the thermograms are shown in Fig. 2 c) were plotted as a function of Na3HB content. As can be seen in Fig. 4, the melting enthalpy increases as the relative content of this hydrated crystalline form in the sample increases at the expense of the solution (lower Na3HB contents) and anhydrous Na3HB (higher contents), respectively. The intersection point of the two linear parts of this dependency occurs at the content of Na3HB equal to 77.5 wt %. Once again, this relative weight content corresponds to the presence of two water molecules per molecule of Na3HB in the crystalline hydrate (Fig. 1 b). The stoichiometry of this crystalline form was hence confirmed by two independent experimental approaches.

Figure 4.

The dependence of the enthalpy of melting of Na3HB dihydrate on concentration.

Glass transitions in Na3HB/water systems

The previous section describes the effect of Na3HB from the viewpoint of equilibrium freezing. On the other hand, freezing of the living organisms usually occurs far from equilibrium, with a great effect of freezing dynamics. As already noted, when determining the equilibrium phase diagram, the most crucial experimental condition was represented by preventing the sample to turn into a glassy state. In contrast, the bioprotective effects of compatible solutes during the freezing or desiccation of an organism are often attributed exactly to this tendency to maintain an amorphous form with water molecules kinetically trapped inside (28,32). Therefore, we have complemented the study with the DSC analysis focused on the state transitions in Na3HB/water systems out of equilibrium.

From the experimental point of view, the specific difference in the DSC procedure lies in the fast cooling of the sample to a temperature well below the glass point before the evaluated heating scan is performed. Hereby, efficient conversion of the sample to the glassy state is assured, preventing the transition of the sample to a thermodynamically favored crystalline phase of Na3HB dihydrate (as it is verified by the absence of the dihydrate melting endotherm peak in the heating scan). The obtained DSC thermograms used for the evaluation of the glass transitions are shown in supporting material (Fig. S3). The composition of the samples significantly influenced their ability to form a glass state. For example, samples with Na3HB concentrations from 6.3 to 37.9 wt % showed both the glass transition step and the ice-melting peak (Fig. S3 b, supporting material). In the zoomed glass transition steps of these thermograms (Fig. S4 c–e), it can also be seen that the glass transition among the analyzed samples varies in temperature and the value of the heat capacity change.

Fig. 5 shows how the glass point and the heat capacity change depend on the Na3HB content. Three distinct concentration regions can be distinguished in both plots shown in Fig. 5. First, samples with Na3HB content below 50 wt % show the coexistence of the glassy state and ice. The composition (i.e., the content of water and Na3HB, respectively) of the glassy state does not change over this region, as indicated by the constant glass transition temperature, while the relative content of the glassy state increases with total Na3HB content in the sample as confirmed by increasing heat capacity change. Similarly, in the samples rich in Na3HB (above 68 wt %), there is a coexistence of a glassy Na3HB/water mixture and crystalline Na3HB phases. Interestingly, it can be seen that the composition of the glassy states of the two regions differs, as can be derived from the border concentration of the two regions (50 and 68 wt %, respectively). While the glassy state in the low-Na3HB region contains seven water molecules per molecule of Na3HB, in the high-Na3HB region this drops to about three water molecules per Na3HB molecule. This again confirms the extraordinary hydrophilicity of Na3HB; for instance, glassy trehalose at ambient temperature is known to contain about one water molecule per glucose ring (i.e., two per one molecule) (28).

Figure 5.

Glass transitions obtained by DSC data: (left) temperature dependence and independence of glass transition on water content; (right) heat capacity of glass transitions.

In contrast, the region of medium concentrations (50–68 wt % Na3HB) differs from the neighboring ones in that the glass temperature increases with Na3HB content while the heat capacity change remains constant. This indicates that these samples are fully in a glassy state. This was confirmed also by the fact that no ice melting was detected in this concentration region. All the water in the sample is restricted in its motion by locking up in the amorphous Na3HB matrix. The disordered state of the Na3HB/water system is prevented from any ordering by too-high energetic barriers of molecular motion, which are hence slowed down below the timescale of the experiment. The range of glass transition temperatures (from about −70°C to −55°C) covered in this concentration region is comparable with the Tg values reported for carbohydrate-based compatible solutes. For instance, a meta-analysis of Tg values of trehalose-water mixtures presented in (25) gives the value of −80°C for a mixture with 40 wt % of water (compare with Tg −60.7°C determined in our study for Na3HB/water mixture with the same water content). Similarly, glass temperatures at equilibrium with ice close to −50°C were reported for sucrose and fructose, whereas it is about −70°C for 3HB as determined in our study (33).

The results of the DSC analysis of the glass transition in the Na3HB/water binary systems were used to construct a state diagram shown in Fig. 6. Again, the cryoprotective effect of Na3HB can be seen in several features of the diagram. Water freezing is reduced partially (below Na3HB content of 40–50 wt %) or completely (above this Na3HB content). Furthermore, in the region where the water freezes, a significant freezing temperature depression is found again.

From the state diagram shown in Fig. 6, it is hardly possible to determine precisely the limiting composition of Na3HB/water mixtures at which the water in the sample stops freezing. For that purpose, the total enthalpies, determined from the corresponding ice-melting endotherms (shown in Fig. S3 b, supporting material), were plotted against the water content in the sample (Fig. 7). From the linear regression and extrapolation to zero enthalpy, the amount of nonfrozen water was determined to be approximately 1.35 g of water per g of Na3HB (or 0.57 g of water per g of sample). Once again, this illustrates the outstanding position of Na3HB among the recognized compatible solutes. For instance, published values of nonfreezing water for sugars range from 0.21 g of water per g of fructose, through 0.26 g of water per g of sucrose, to 0.31 g of water per gram of trehalose (34).

Figure 7.

Amount of nonfreezing water per gram of Na3HB obtained by DSC data as a function of water content on enthalpy of melting of water. To see this figure in color, go online.

Biological implications

Generally, 3HB is a very important biomolecule that can be found in numerous biological systems in which 3HB fulfills various functions. 3HB is an intermediate precursor of various metabolite pathways, such as the metabolism of leucine in humans. More importantly, in humans and other higher organisms, 3HB is synthesized in the liver, circulates in the blood, and, along with glucose and other ketone bodies, serves as an energy fuel for cells. The concentration of 3HB in the blood of adults varies between 5 and 335 μmol/L, and notably higher concentrations (1–2 mmol/L) occur in individuals experiencing ketosis (35). A substantially higher concentration of 3HB can be found in prokaryotes capable of PHB accumulation, such as Cupriavidus necator (17) or Rhodospirillum rubrum (36). Since the PHB metabolism constitutes simultaneous synthesis and hydrolysis of PHB granules by the action of PHB granule-associated enzymes PHB synthase and PHB depolymerase, the intracellular concentration of 3HB reaches about 100 mmol/L (12,13), which is about 16.5-fold higher than 3HB concentration in PHB non-accumulating mutant bacterial strain (12). The intracellular concentration of 3HB is comparable with the cell contents of compatible solutes such as glycerol, trehalose, or ectoines, which are accumulated in the cell under stress conditions.

Similar to other compatible solutes, 3HB also reveals a general protective function: it is capable of shielding biomolecules from denaturation induced by high temperature or oxidation from concentrations of 50 or 100 mmol/L (12). Moreover, in the follow-up study (14), it was also observed that Na3HB acts as a potent cryoprotectant that could protect various biological systems when exposed to repeated freezing and thawing. When Na3HB was applied at the concentration of 100 mmol/L, it protected the model enzyme from losing activity during seven subsequent freezing/thawing cycles, and its protective effect was even slightly higher than that observed in trehalose. Similarly, when Na3HB was added to a final concentration of 100 mmol/L to the suspension of yeast Saccharomyces cerevisiae in the same study (14), it also protected the cells against repeated freezing and thawing even more efficiently than well-recognized cryoprotectants such as trehalose, ectoine, or glycerol. The cryoprotective potential of Na3HB is also confirmed by the results of the current thermodynamic study, whether from the point of view of the position of the eutectic point (the lowest achievable melting temperature), depression of the ice temperature for the lower Na3HB concentrations, the unique glass transitions behavior, or the high amount of nonfrozen water in the studied Na3HB/water systems.

The fact that Na3HB represents a very potent cryoprotectant is not only of fundamental interest concerning its biological importance, especially in PHB-accumulating prokaryotes, but it might have also numerous applications. For instance, trehalose and glycerol are frequently used as protectants for cryopreservation of various biological samples; e.g., viable microbial or cellular cultures, proteins, nucleic acids, antibodies, and other biologically active substances (37). Our results suggest that Na3HB could be advantageously used in the same manner. Moreover, since numerous cryoprotectants are also used to maintain the activity of various biological samples during and after freeze-drying, it would also be interesting to test the protective potential of Na3HB in this regard. Considering all the fundamental physicochemical properties of Na3HB determined in this work, it seems that Na3HB possesses all the prerequisites to be used also as a lyoprotectant in freeze-drying. Further, various cryoprotectants are widely used in food preservation to protect food from undesirable changes in texture, taste, and overall acceptability upon freezing, frozen storage, and subsequent thawing (38,39). Considering all the qualities of Na3HB described in this work and also the facts that Na3HB is a safe substance naturally present in the human body in high concentrations and that it is a sensorially neutral substance (i.e., not influencing the natural flavor and taste of foods), it could be used as a food additive to improve the quality and stability of frozen foodstuffs. Last but not least, Na3HB can be relatively simply produced biotechnologically or by chemical or enzymatical hydrolysis of PHB (40,41); therefore, it is an easily available and relatively cheap substance able to compete with well-established cryopreservatives.

Conclusions

The study emphasizes the fundamental properties of Na3HB, ubiquitous biological molecule that provides numerous biological functions. This work was focused on the characterization of Na3HB concerning its important cryoprotective function. Na3HB demonstrates an extraordinary affinity for water that is at least comparable with the other known compatible solutes such as trehalose. Furthermore, the data demonstrated that Na3HB effectively decreases the freezing point of water solutions and provides a large amount of nonfrozen water. These results are of fundamental importance for instance concerning the explanation of the widespread capability of PHB synthesis among psychrophilic bacteria, but this study might also open a gate for the application of Na3HB in the food industry or biotechnology as a potent cryoprotectant or lyoprotectant. These potential applications are the focus of follow-up studies.

Author contributions

E.S., investigation, data curation, and writing – original draft preparation; S.O., writing – review and editing, supervision, funding acquisition; V.K., conceptualization, investigation, validation, methodology, supervision, writing – review and editing, and project administration; P.S., conceptualization, methodology, writing – original draft, writing – review and editing, supervision, and project administration.

Editor: Heiko Heerklotz.