High pressures depress the onset of intracellular vitrification

Stewart Gault; Charles S. Cockell

doi:10.1371/journal.pone.0339165

Abstract

The low temperature limit for life remains elusive and poorly understood. This ignorance is further compounded when applied to life in multi-extreme environments where low temperatures combine with factors such as high salt concentrations, or high environmental pressures. It has been proposed that the onset of intracellular vitrification enforces a biophysical low temperature limit for unicellular life at ~ −23 °C. However, it has not been demonstrated how high-pressures affect intracellular vitrification, which is vital for understanding the habitability of low temperature, subsurface environments, both on Earth and on other planetary bodies. Here, we used high-pressure differential scanning calorimetry to measure the intracellular vitrification of Bacillus subtilis across pressures ranging from 1 to 1000 bar. We find that high pressures depress the onset of intracellular vitrification in a pressure dependent manner, which is tightly correlated with the ability of pressure to depress the freezing point of water. Additionally, we show that sub-molar concentrations of NaCl can act in combination with high pressures to further depress intracellular vitrification, highlighting the interplay between temperature, pressure, and ions in influencing the physical state of cells in natural environments. These results show that cells in subzero high-pressure environments can remain liquidous, and potentially metabolically active, and not merely vitrified and preserved. Additionally, our results provide considerations in the preparation of biological samples through high-pressure freezing for electron microscopy, particularly those associated with high concentrations of cryoprotectants.

Citation: Gault S, Cockell CS (2025) High pressures depress the onset of intracellular vitrification. PLoS One 20(12): e0339165. https://doi.org/10.1371/journal.pone.0339165

Editor: Barry L. Bentley, Cardiff Metropolitan University, UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND

Received: August 7, 2025; Accepted: December 2, 2025; Published: December 18, 2025

Copyright: © 2025 Gault, Cockell. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The potential for active microbial activity in high-pressure subzero environments remains poorly understood. On Earth, such extreme environments are typified by Lake Vostok in Antarctica, or deep subsurface permafrost in Siberia. Lake Vostok is characterised by pressures of ~400 bar due to 4 km of overlaying ice, and temperatures of approximately −3 °C [1]. In Siberia, particularly the Lena and Yana river basins, modelling suggests that the permafrost can extend to depths of ~1.5 km, experiencing pressures over 100 bar, with environmental temperatures just below 0 °C [2]. The inaccessibility of these multi-extreme environments has significantly hampered our understanding of life’s ability to exist under such extremes. In contrast we know that near-surface subzero environments contain a variety of psychrophiles and cryophiles [3], with isolated species such as Planococcus halocryophilus remaining metabolically active at temperatures as low as −25 °C [4].

In addition to the terrestrial deep subsurface, evidence suggests that planetary bodies beyond Earth harbour subzero subterranean aqueous environments. On Mars, water is either predicted to exist as deep groundwater [5], or as subglacial lakes [6,7] where temperatures would be on the order of −70 °C, although the latter phenomena remains controversial. Further afield, the icy moons Europa, Enceladus, and Titan contain liquid water oceans trapped beneath their icy crusts. The icy moon oceans span a range of temperatures and pressures, with Europa and Enceladus hosting oceans at ~0 °C, but with ocean bottom pressures of 2000 bar [8] and <100 bar [9–11] respectively. Titan’s ocean however is thought to have a much broader temperature and pressure profile, with the upper water-ice boundary thought to ~1500 bar and −14 °C, whereas the ocean bottom water-rock/ice interface is thought to be ~ 7500 bar and −1 °C [12].

To understand the potential for such extreme environments to host life, or its remnants, we must first understand the limits to life in high-pressure, subzero environments. Attempts have been made to sample the microbial diversity of Lake Vostok, though initial measurements were significantly hampered by contamination from drill fluids [13]. However, sampling of a shallower Antarctic subglacial lake, Lake Whillans, revealed a diverse microbial ecology [14–16]. Ultimately though our perception of the limits to life in high pressure subzero temperature environments remains significantly limited.

In the absence of in situ measurements, the field of microbial biophysics offers a window into this otherwise challenging area of multi-extreme microbiology. From the biophysical perspective, the low temperature limit for life is thought to be enforced by the onset of intracellular vitrification, as was proposed by Clarke et al [17]. Intracellular vitrification occurs when cells lose their intracellular water through osmosis as the freezing of the extracellular environment concentrates extracellular solutes. The loss of intracellular water continues until a point is reached at which intracellular diffusion ceases and a dramatic increase in viscosity occurs. Mechanistically, the process of intracellular vitrification is akin to the glass transition, T_g′ of maximally freeze-concentrated protein solutions [18]. The intracellular vitrification of unicellular organisms occurs at approximately −23 °C in the absence of salts and other substances which can depress the freezing point of water. This therefore suggests that tentative reports of microbial activity at temperatures below −30 °C are reliant on the depression of intracellular vitrification [19]. Cryoprotectants such as glycerol, either delivered exogenously [20] or produced in vivo [21] can significantly depress intracellular vitrification, as can the presence of environmentally relevant salts such as Mg(ClO₄)₂, which at 2.5 M can depress Bacillus subtilis’ (B. subtilis) intracellular vitrification to −83 °C [22]. While these studies show that the biophysical limits to life in subzero environments can be shifted by the environmental chemical composition, the effect of high pressures, relevant to Earth’s deep cryosphere and icy moon oceans, remains unknown.

To address this terra incognita, we have utilised high-pressure differential scanning calorimetry (HPDSC) to measure the effect of pressures up to 1 kbar on the intracellular vitrification of B. subtilis. These results will help elucidate the potential for life and active biochemistry in high-pressure, subzero environments and assist in the interpretation of data from missions assessing the habitability of environments beyond Earth.

Materials and methods

Bacillus subtilis DSM 10 was obtained from the Leibniz Institute, DSMZ

Cell culturing and sample preparation

B. subtilis was cultured overnight in 50 mL of growth media (5 g/L peptone, 3 g/ L meat extract, pH 7, 25°C) in three separate flasks. The separate cell suspensions were then pelleted at 3500 g for 10 minutes, the supernatant was discarded, and the pellets were resuspended in deionized water then combined into a single cell suspension. This concentrated cell suspension was pelleted and washed twice more with deionized water. For experiments investigating the effect of NaCl on vitrification, the washed cell pellet was resuspended in the desired NaCl concentration for 1 hour before being pelleted as before. To remove excess solution from the final cell pellet, the cells were centrifuged a final time at 10,000 g for 1 minute.

High pressure differential scanning calorimetry

High-pressure differential scanning calorimetry was conducted using a Microcalvet calorimeter with Hastelloy high-pressure cells, manufactured by Setaram with a circulating water bath to cool the HPDSC Peltier. High pressures were generated using a Teledyne LABS Syrixus 65x syringe pump with N₂ as the pressurising gas.

For HPDSC measurements, 15–40 mg of B. subtilis cell pellets were loaded into an aluminium cup weighing 73 mg, 6 mm in height and 5 mm in diameter with walls 0.25 mm thick. The aluminium cup containing the cell pellet was placed inside the sample chamber with an identical empty aluminium cup placed inside the reference chamber. For the temperature ramps, cells were held isothermally for 5 minutes at 10 °C, before being cooled to –45 °C at 1 °C/min, held isothermally at –45 °C for 10 minutes, then heated to 10 °C at 1 °C/min. The HPDSC scans were conducted at 1, 250, 500, 750, and 1000 bar. Measurements for each pressure step were taken on multiple and separate days, using fresh samples grown from the same cryogenic stock.

The HPDSC heating scan traces were analysed with TRIOS software (TA Instruments, Version 5.1). For analysis, the first derivative of the heating scan was produced and smoothed to 200 neighbours to highlight the intracellular vitrification signal. The vitrification signal was then analysed by recording the temperatures of the onset, peak, and end, denoted T_onset, T_peak, and T_end respectively [22]. T_onset was taken as the tangent between the flat baseline prior to the vitrification signal and the steepest portion of the initial inflection. T_peak corresponded to the maximum height of the vitrification signal, while T_end was measured as the tangent between the descending limb of the vitrification signal and the upwards inflection of the water melting peak.

Results and discussion

Here we report the first experimental investigation of the effect of isobaric high pressures on microbial intracellular vitrification. Fig 1 shows the heating scans of the B. subtilis cell pellets across increasing pressures. From Fig 1 the pressure dependent decrease in the melting temperature of the extracellular ice is readily apparent. The clear truncation of the ice melting signal is due to saturation of the DSC, and results from the trade-offs between the sensitivity of the DSC utilised and the sample mass required to observe intracellular vitrification signal. The incomplete nature of the ice melting peak prohibits a robust thermodynamic analysis of the melting curves, however the intracellular vitrification signal is still readily separated from the derivative of the heating scans. Fig 2 shows the temperatures at which the T_onset, T_peak, and T_end of the intracellular vitrification signal was recorded across pressure. At 1 bar, B. subtilis cell pellets exhibited a mean T_peak of –24.11 °C, which is consistent with previous vitrification values for B. subtilis and other unicellular organisms [17]. As the pressure was increased, a pressure dependent depression of intracellular vitrification was observed. By 1000 bar the mean T_peak had been depressed to –29.78 °C, with 250, 500, and 750 bar depressing T_peak to −25.30, −26.82, and −29.02 °C respectively. A simple linear regression of T_onset, T_peak, and T_end across pressure suggested their slopes are significantly non-zero, with p values of 0.0005, 0.0003, and 0.0006 respectively (all statistical parameters are provided within S1 Dataset).

Download:

Fig 1. DSC heat scans of B. subtilis across pressure.

The heating scans of B. subtilis cell pellets across increasing pressures from 1 bar (blue, n = 5), 250 bar (red, n = 5), 500 bar (green, n = 4), 750 bar (purple, n = 5), and 1000 bar (orange, n = 5). Traces have been offset for visual clarity.

https://doi.org/10.1371/journal.pone.0339165.g001

Download:

Fig 2. Intracellular vitrification of Bacillus subtilis across pressure.

The T_onset (blue squares), T_peak (red triangles), and T_end (green triangles), of the B. subtilis intracellular vitrification signal at 1 bar (n = 5), 250 bar (n = 5), 500 bar (n = 4), 750 bar (n = 5), and 1000 bar (n = 5) and their associated linear regressions.

https://doi.org/10.1371/journal.pone.0339165.g002

The gradual depression of intracellular vitrification by pressure is consistent with the pressure induced depression of water’s freezing point. This reinforces the causal relationship between the freezing of the extracellular environment and the onset of intracellular vitrification, as stipulated by Clarke et al. [17]. Our results support the hypothesis that the measured intracellular glass transition is mechanistically identical to the glass transition or colloid transition T_g′, of maximally freeze-concentrated protein solutions [18]. The T_g′ of protein solutions tends to occur around −10 °C [18], with the addition of small molecular weight excipients (such as salts or sugars) lowering the onset of T_g′. The onset of intracellular vitrification at ambient pressures at lower temperatures of ~−23 °C is therefore consistent with the fact that cells are predominantly protein solutions mixed with a diverse array of small molecules which depress intracellular vitrification from −10 °C that would be expected if cells were purely proteinaceous in nature. As the T_g′ of protein solutions is essentially an effect of cryoconcentration, this explains that the pressure induced depression of intracellular vitrification is due to the inhibition of ice crystallisation in keeping with water’s phase diagram. In contrast, previous reports have shown that the application of pressure increases the T_g of halide solutions [23], which further suggests that the glass transition we observe in our data is mechanistically distinct from the glass transition of saline solutions or cryoprotectant solutions. This highlights the care that must be taken when discussing glass transitions in complex samples (such as whole cells), as multiple physical events can give rise to the characteristic glass transition observed via DSC and so it is important to delineate between the mechanisms underpinning the transitions being discussed.

It has been previously shown that salts can depress the T_g of simple sugar solutions [24] and of whole cells [22]. As real environments are typically expected to contain salts, which themselves can depress the freezing point of water, we expected that the presence of salt and pressure should have a combined effect on intracellular vitrification. We demonstrate this in Fig 3 by showing that the presence of NaCl and high pressures can exhibit a combined effect on the depression of intracellular vitrification. From the representative data, at 1 bar, 0.25 M and 0.5 M NaCl depressed intracellular vitrification to ~−24 and −32 °C respectively. Increasing the pressure to 1000 bar depressed intracellular vitrification at 0 M and 0.25 M NaCl by −7 °C, while a minor shift was observed in the presence of 0.5 M NaCl, with the vitrification peak being depressed by −3 °C. These results suggest that the intracellular vitrification of unicellular organisms will occur at temperatures below the liquid-solid phase boundary of an environmentally relevant brine’s temperature-pressure-salt phase diagram. This is likely due to the high concentration of organic molecules within cells, and the nanoconfined nature of intracellular water [25], which may act in unison to further modulate the phase behaviour of a cell’s intracellular contents.

Download:

Fig 3. B. subtilis heating scans across pressure and NaCl concentrations: Derivatives of heating scans of B. subtilis pellets showing the vitrification signal at 1 bar (solid lines), and 1000 bar (dashed lines) and 0 M (blue), 0.25 M (red), and 0.5 M (green) NaCl.

Each curve represents a single measurement.

https://doi.org/10.1371/journal.pone.0339165.g003

Due to the saturation of the heat signal caused by the mass of water in the sample required to measure vitrification, we are unable to provide a quantitative thermodynamic analysis of the melting and freezing of the water within our samples. The relatively large sample mass meant that the freezing/melting signal of water was consistently saturating the calorimeter, however qualitatively it was observed that the freezing and melting of water was depressed by high pressures and salt concentrations, as would be expected with their phase diagrams.

These results suggest that unicellular organisms in subzero high-pressure brines would remain in a fluid state under environmental conditions analogous to those experienced in the ice surrounding Lake Vostok, deep subsurface permafrost, and the ice shells of Europa and Enceladus, close to the ocean-ice boundary. However, our conceptual understanding of whether an environment is habitable or not is extremely biased by our understanding of the biophysical and biochemical limits to terrestrial life. So, while Lake Vostok is habitable from our theoretical understanding of life’s biophysical limits, it is yet to be demonstrated whether microbial life itself can remain active in these cold, high-pressure, nutrient poor environments. In the context of high pressure, subzero aqueous environments like those of Mars, Europa, Enceladus, and Titan, the lack of representative, accessible terrestrial analogues to these multi-extreme environments further compounds our ignorance as to their habitability.

As the microcalorimeter’s highest operating pressure is 1 kbar we were unable explore the pressures that are expected at the base of Europa and Titan’s oceans, 2 kbar and 7.5 kbar respectively [8,12]. The potential for brine veins in high-pressure ice to support life is further tempered by our understanding of the relationship between pressure and biochemical structure. Pressures of 7.5 kbar are sufficient to denature proteins [26,27], as such, life in these environments would necessitate biochemical adaptations and innovations that life on Earth has not experienced in its evolutionary history. The high-pressure DSC’s lower operational temperature of −45 °C also precluded examining environmental conditions analogous to the Martain subsurface where pressures are expected to be < 1 kbar, but the temperature can be as low as −70 °C. Such data would have complimented previous work where we showed that molar concentrations of Mg(ClO₄)₂ can significantly depress B. subtilis’ vitrification, with 2.5M Mg(ClO₄)₂ depressing T_peak to ~−83 °C. Given that intracellular vitrification is closely linked to the freezing points of aqueous solutions, fully mapping the high-pressure phase diagrams of environmentally relevant ionic solutions will greatly expand our understanding of where the biophysical low temperature limits for life may be found in extreme environments in our solar system.

Our results may also impact protocol design for electron microscopy sample preparation. High-pressure vitrification and freezing is a mainstay for the cryofixation of electron microscopy samples, wherein small volumes of samples are sealed then rapidly cooled [28]. As the water within the sample begins to freeze, the ensuing volumetric expansion causes a rapid increase in the internal pressure of the sample which inhibits ice crystallisation, instead inducing the formation of amorphously frozen/ vitrified water, allowing for greater preservation of biological structures. Until now however it had not been demonstrated what effect high pressures have on cellular vitrification. Therefore our results suggest that the process of high-pressure freezing will not only depress ice crystal formation, but may also affect the temperature at which the cells themselves undergo a glass transition. Given the demonstrated relationship between pressure and ion composition on intracellular vitrification, it stands that high concentrations of deeply eutectic ions may affect the degree to which samples can fully vitrify, thus affecting sample quality for electron microscopy. This would likely affect the sample preparation of halophiles which require high salt concentrations for optimal growth, or species which can produce high concentrations of cryoprotectants in vivo [21], which may need to be brought to lower temperatures to achieve a sufficient degree of vitrification.

Supporting information

S1 Dataset. Raw data for manuscript figures and statistical analysis.

https://doi.org/10.1371/journal.pone.0339165.s001

(XLSX)

Acknowledgments

I would like to thank Dr Claire Hobday and Dr Joshua Levinsky for providing access to and training for the high-pressure differential scanning calorimeter and Toby Fu for machining the aluminium cup.

References

1. Robin GDQ, Drewry DJ, Meldrum DT. International studies of ice sheet and bedrock. Philosophical Transactions of the Royal Society of London B, Biological Sciences. 1997;279: 185–196.
- View Article
- Google Scholar
2. Schuur EAG, Bockheim J, Canadell JG, Euskirchen E, Field CB, Goryachkin SV, et al. Vulnerability of permafrost carbon to climate change: implications for the global carbon cycle. BioScience. 2008;58(8):701–14.
- View Article
- Google Scholar
3. Steven B, Léveillé R, Pollard WH, Whyte LG. Microbial ecology and biodiversity in permafrost. Extremophiles. 2006;10(4):259–67. pmid:16550305
- View Article
- PubMed/NCBI
- Google Scholar
4. Mykytczuk NCS, Foote SJ, Omelon CR, Southam G, Greer CW, Whyte LG. Bacterial growth at -15 °C; molecular insights from the permafrost bacterium Planococcus halocryophilus Or1. ISME J. 2013;7(6):1211–26. https://doi.org/10.1038/ismej.2013.8 pmid:23389107
5. Clifford SM, Lasue J, Heggy E, Boisson J, McGovern P, Max MD. Depth of the Martian cryosphere: Revised estimates and implications for the existence and detection of subpermafrost groundwater. J Geophys Res. 2010;115(E7).
- View Article
- Google Scholar
6. Orosei R, Lauro SE, Pettinelli E, Cicchetti A, Coradini M, Cosciotti B, et al. Radar evidence of subglacial liquid water on Mars. Science. 2018;361(6401):490–3. https://doi.org/10.1126/science.aar7268 pmid:30045881
7. Lauro SE, Pettinelli E, Caprarelli G, Guallini L, Rossi AP, Mattei E, et al. Multiple subglacial water bodies below the south pole of Mars unveiled by new MARSIS data. Nat Astron. 2020;5(1):63–70.
- View Article
- Google Scholar
8. Naganuma T, Uematsu H. Dive Europa: a search-for-life initiative. Biol Sci Space. 1998;12(2):126–30. pmid:11541880
- View Article
- PubMed/NCBI
- Google Scholar
9. Taubner R-S, Leitner JJ, Firneis MG, Hitzenberger R. Modelling the Interior Structure of Enceladus Based on the 2014’s Cassini Gravity Data. Orig Life Evol Biosph. 2016;46(2–3):283–8. https://doi.org/10.1007/s11084-015-9475-9 pmid:26559966
10. Iess L, Stevenson DJ, Parisi M, Hemingway D, Jacobson RA, Lunine JI, et al. The gravity field and interior structure of Enceladus. Science. 2014;344(6179):78–80. https://doi.org/10.1126/science.1250551 pmid:24700854
11. Thomas PC, Tajeddine R, Tiscareno MS, Burns JA, Joseph J, Loredo TJ, et al. Enceladus’s measured physical libration requires a global subsurface ocean. Icarus. 2016;264:37–47.
- View Article
- Google Scholar
12. Meyer-Dombard DR, Malas J, Russo DC, Kenig F. Astrobiology of Titan’s subglacial, high pressure ocean: A review and introduction of a relevant experimental system. Titan After Cassini-Huygens. Elsevier. 2025. p. 423–71.
- View Article
- Google Scholar
13. Michaud AB, Vick-Majors TJ, Achberger AM, Skidmore ML, Christner BC, Tranter M, et al. Environmentally clean access to Antarctic subglacial aquatic environments. Antarctic Science. 2020;32(5):329–40.
- View Article
- Google Scholar
14. Vick-Majors TJ, Achberger A, Santibáñez P, Dore JE, Hodson T, Michaud AB, et al. Biogeochemistry and microbial diversity in the marine cavity beneath the McMurdo Ice Shelf, Antarctica. Limnol Oceanogr. 2015;61(2):572–86.
- View Article
- Google Scholar
15. Vick-Majors TJ, Mitchell AC, Achberger AM, Christner BC, Dore JE, Michaud AB, et al. Physiological Ecology of Microorganisms in Subglacial Lake Whillans. Front Microbiol. 2016;7:1705. pmid:27833599
- View Article
- PubMed/NCBI
- Google Scholar
16. Michaud AB, Vick-Majors TJ, Achberger AM, Skidmore ML, Christner BC, Tranter M, et al. Environmentally clean access to Antarctic subglacial aquatic environments. Antarctic Science. 2020;32(5):329–40.
- View Article
- Google Scholar
17. Clarke A, Morris GJ, Fonseca F, Murray BJ, Acton E, Price HC. A Low Temperature Limit for Life on Earth. PLoS One. 2013;8(6):e66207. pmid:23840425
- View Article
- PubMed/NCBI
- Google Scholar
18. Chang BS, Randall CS. Use of subambient thermal analysis to optimize protein lyophilization. Cryobiology. 1992;29(5):632–56.
- View Article
- Google Scholar
19. Panikov NS, Flanagan PW, Oechel WC, Mastepanov MA, Christensen TR. Microbial activity in soils frozen to below −39 °C. Soil Biology and Biochemistry. 2006;38(4):785–94.
- View Article
- Google Scholar
20. Fonseca F, Meneghel J, Cenard S, Passot S, Morris GJ. Determination of Intracellular Vitrification Temperatures for Unicellular Micro Organisms under Conditions Relevant for Cryopreservation. PLoS One. 2016;11(4):e0152939. pmid:27055246
- View Article
- PubMed/NCBI
- Google Scholar
21. Sformo T, Walters K, Jeannet K, Wowk B, Fahy GM, Barnes BM, et al. Deep supercooling, vitrification and limited survival to -100{degrees}C in the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) larvae. J Exp Biol. 2010;213(3):502–9. pmid:20086136
- View Article
- PubMed/NCBI
- Google Scholar
22. Gault S, Fonseca F, Cockell CS. Preservation of Bacillus subtilis’ cellular liquid state at deep sub-zero temperatures in perchlorate brines. Commun Biol. 2024;7(1):588. https://doi.org/10.1038/s42003-024-06277-4 pmid:38755264
23. Kanno H, Angell CA. Homogeneous nucleation and glass formation in aqueous alkali halide solutions at high pressures. J Phys Chem. 1977;81(26):2639–43.
- View Article
- Google Scholar
24. Her LM, Deras M, Nail SL. Electrolyte-induced changes in glass transition temperatures of freeze-concentrated solutes. Pharm Res. 1995;12(5):768–72. pmid:7479566
- View Article
- PubMed/NCBI
- Google Scholar
25. Yao Y, Fella V, Huang W, Zhang KAI, Landfester K, Butt H-J, et al. Crystallization and Dynamics of Water Confined in Model Mesoporous Silica Particles: Two Ice Nuclei and Two Fractions of Water. Langmuir. 2019;35(17):5890–901. pmid:30946592
- View Article
- PubMed/NCBI
- Google Scholar
26. Gault S, Jaworek MW, Winter R, Cockell CS. High pressures increase α-chymotrypsin enzyme activity under perchlorate stress. Commun Biol. 2020;3(1):550. pmid:33009512
- View Article
- PubMed/NCBI
- Google Scholar
27. Knop J-M, Mukherjee S, Jaworek MW, Kriegler S, Manisegaran M, Fetahaj Z, et al. Life in Multi-Extreme Environments: Brines, Osmotic and Hydrostatic Pressure─A Physicochemical View. Chem Rev. 2023;123(1):73–104. pmid:36260784
- View Article
- PubMed/NCBI
- Google Scholar
28. Leunissen JLM, Yi H. Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy. J Microsc. 2009;235(1):25–35. pmid:19566624
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Robin GDQ, Drewry DJ, Meldrum DT. International studies of ice sheet and bedrock. Philosophical Transactions of the Royal Society of London B, Biological Sciences. 1997;279: 185–196.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Schuur EAG, Bockheim J, Canadell JG, Euskirchen E, Field CB, Goryachkin SV, et al. Vulnerability of permafrost carbon to climate change: implications for the global carbon cycle. BioScience. 2008;58(8):701–14.
View Article
Google Scholar

[5] View Article

[6] Google Scholar

[ref3] 3. Steven B, Léveillé R, Pollard WH, Whyte LG. Microbial ecology and biodiversity in permafrost. Extremophiles. 2006;10(4):259–67. pmid:16550305
View Article
PubMed/NCBI
Google Scholar

[8] View Article

[9] PubMed/NCBI

[10] Google Scholar

[ref4] 4. Mykytczuk NCS, Foote SJ, Omelon CR, Southam G, Greer CW, Whyte LG. Bacterial growth at -15 °C; molecular insights from the permafrost bacterium Planococcus halocryophilus Or1. ISME J. 2013;7(6):1211–26. https://doi.org/10.1038/ismej.2013.8 pmid:23389107

[ref5] 5. Clifford SM, Lasue J, Heggy E, Boisson J, McGovern P, Max MD. Depth of the Martian cryosphere: Revised estimates and implications for the existence and detection of subpermafrost groundwater. J Geophys Res. 2010;115(E7).
View Article
Google Scholar

[13] View Article

[14] Google Scholar

[ref6] 6. Orosei R, Lauro SE, Pettinelli E, Cicchetti A, Coradini M, Cosciotti B, et al. Radar evidence of subglacial liquid water on Mars. Science. 2018;361(6401):490–3. https://doi.org/10.1126/science.aar7268 pmid:30045881

[ref7] 7. Lauro SE, Pettinelli E, Caprarelli G, Guallini L, Rossi AP, Mattei E, et al. Multiple subglacial water bodies below the south pole of Mars unveiled by new MARSIS data. Nat Astron. 2020;5(1):63–70.
View Article
Google Scholar

[17] View Article

[18] Google Scholar

[ref8] 8. Naganuma T, Uematsu H. Dive Europa: a search-for-life initiative. Biol Sci Space. 1998;12(2):126–30. pmid:11541880
View Article
PubMed/NCBI
Google Scholar

[20] View Article

[21] PubMed/NCBI

[22] Google Scholar

[ref9] 9. Taubner R-S, Leitner JJ, Firneis MG, Hitzenberger R. Modelling the Interior Structure of Enceladus Based on the 2014’s Cassini Gravity Data. Orig Life Evol Biosph. 2016;46(2–3):283–8. https://doi.org/10.1007/s11084-015-9475-9 pmid:26559966

[ref10] 10. Iess L, Stevenson DJ, Parisi M, Hemingway D, Jacobson RA, Lunine JI, et al. The gravity field and interior structure of Enceladus. Science. 2014;344(6179):78–80. https://doi.org/10.1126/science.1250551 pmid:24700854

[ref11] 11. Thomas PC, Tajeddine R, Tiscareno MS, Burns JA, Joseph J, Loredo TJ, et al. Enceladus’s measured physical libration requires a global subsurface ocean. Icarus. 2016;264:37–47.
View Article
Google Scholar

[26] View Article

[27] Google Scholar

[ref12] 12. Meyer-Dombard DR, Malas J, Russo DC, Kenig F. Astrobiology of Titan’s subglacial, high pressure ocean: A review and introduction of a relevant experimental system. Titan After Cassini-Huygens. Elsevier. 2025. p. 423–71.
View Article
Google Scholar

[29] View Article

[30] Google Scholar

[ref13] 13. Michaud AB, Vick-Majors TJ, Achberger AM, Skidmore ML, Christner BC, Tranter M, et al. Environmentally clean access to Antarctic subglacial aquatic environments. Antarctic Science. 2020;32(5):329–40.
View Article
Google Scholar

[32] View Article

[33] Google Scholar

[ref14] 14. Vick-Majors TJ, Achberger A, Santibáñez P, Dore JE, Hodson T, Michaud AB, et al. Biogeochemistry and microbial diversity in the marine cavity beneath the McMurdo Ice Shelf, Antarctica. Limnol Oceanogr. 2015;61(2):572–86.
View Article
Google Scholar

[35] View Article

[36] Google Scholar

[ref15] 15. Vick-Majors TJ, Mitchell AC, Achberger AM, Christner BC, Dore JE, Michaud AB, et al. Physiological Ecology of Microorganisms in Subglacial Lake Whillans. Front Microbiol. 2016;7:1705. pmid:27833599
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref16] 16. Michaud AB, Vick-Majors TJ, Achberger AM, Skidmore ML, Christner BC, Tranter M, et al. Environmentally clean access to Antarctic subglacial aquatic environments. Antarctic Science. 2020;32(5):329–40.
View Article
Google Scholar

[42] View Article

[43] Google Scholar

[ref17] 17. Clarke A, Morris GJ, Fonseca F, Murray BJ, Acton E, Price HC. A Low Temperature Limit for Life on Earth. PLoS One. 2013;8(6):e66207. pmid:23840425
View Article
PubMed/NCBI
Google Scholar

[45] View Article

[46] PubMed/NCBI

[47] Google Scholar

[ref18] 18. Chang BS, Randall CS. Use of subambient thermal analysis to optimize protein lyophilization. Cryobiology. 1992;29(5):632–56.
View Article
Google Scholar

[49] View Article

[50] Google Scholar

[ref19] 19. Panikov NS, Flanagan PW, Oechel WC, Mastepanov MA, Christensen TR. Microbial activity in soils frozen to below −39 °C. Soil Biology and Biochemistry. 2006;38(4):785–94.
View Article
Google Scholar

[52] View Article

[53] Google Scholar

[ref20] 20. Fonseca F, Meneghel J, Cenard S, Passot S, Morris GJ. Determination of Intracellular Vitrification Temperatures for Unicellular Micro Organisms under Conditions Relevant for Cryopreservation. PLoS One. 2016;11(4):e0152939. pmid:27055246
View Article
PubMed/NCBI
Google Scholar

[55] View Article

[56] PubMed/NCBI

[57] Google Scholar

[ref21] 21. Sformo T, Walters K, Jeannet K, Wowk B, Fahy GM, Barnes BM, et al. Deep supercooling, vitrification and limited survival to -100{degrees}C in the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) larvae. J Exp Biol. 2010;213(3):502–9. pmid:20086136
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref22] 22. Gault S, Fonseca F, Cockell CS. Preservation of Bacillus subtilis’ cellular liquid state at deep sub-zero temperatures in perchlorate brines. Commun Biol. 2024;7(1):588. https://doi.org/10.1038/s42003-024-06277-4 pmid:38755264

[ref23] 23. Kanno H, Angell CA. Homogeneous nucleation and glass formation in aqueous alkali halide solutions at high pressures. J Phys Chem. 1977;81(26):2639–43.
View Article
Google Scholar

[64] View Article

[65] Google Scholar

[ref24] 24. Her LM, Deras M, Nail SL. Electrolyte-induced changes in glass transition temperatures of freeze-concentrated solutes. Pharm Res. 1995;12(5):768–72. pmid:7479566
View Article
PubMed/NCBI
Google Scholar

[67] View Article

[68] PubMed/NCBI

[69] Google Scholar

[ref25] 25. Yao Y, Fella V, Huang W, Zhang KAI, Landfester K, Butt H-J, et al. Crystallization and Dynamics of Water Confined in Model Mesoporous Silica Particles: Two Ice Nuclei and Two Fractions of Water. Langmuir. 2019;35(17):5890–901. pmid:30946592
View Article
PubMed/NCBI
Google Scholar

[71] View Article

[72] PubMed/NCBI

[73] Google Scholar

[ref26] 26. Gault S, Jaworek MW, Winter R, Cockell CS. High pressures increase α-chymotrypsin enzyme activity under perchlorate stress. Commun Biol. 2020;3(1):550. pmid:33009512
View Article
PubMed/NCBI
Google Scholar

[75] View Article

[76] PubMed/NCBI

[77] Google Scholar

[ref27] 27. Knop J-M, Mukherjee S, Jaworek MW, Kriegler S, Manisegaran M, Fetahaj Z, et al. Life in Multi-Extreme Environments: Brines, Osmotic and Hydrostatic Pressure─A Physicochemical View. Chem Rev. 2023;123(1):73–104. pmid:36260784
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref28] 28. Leunissen JLM, Yi H. Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy. J Microsc. 2009;235(1):25–35. pmid:19566624
View Article
PubMed/NCBI
Google Scholar

[83] View Article

[84] PubMed/NCBI

[85] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Cell culturing and sample preparation

High pressure differential scanning calorimetry

Results and discussion

Supporting information

S1 Dataset. Raw data for manuscript figures and statistical analysis.

Acknowledgments

References