Introduction

Given the fact that water is one of the principal ingredients of cellular life (Daniel et al., 2004), insights into the minimum water requirements of cells are imperative to understanding both the functionality of living systems (at every level, from biomacromolecule to biosphere) and the origins of life itself. The generally held opinion is that life appeared independently on Earth and, possibly, elsewhere in the Solar System (Clancy et al., 2005), though one other explanation for the presence of life on Earth is that it appeared on another planet and was transported here in the form of prokaryotes or their ancestors (an idea known as panspermia; Thomson, 1871). Until recently, eukaryotic microbes have held the record for life under water‐constrained conditions, as some species are capable of cell division down to a water activity (a w )1 of 0.605 at high sugar concentrations (Pitt and Christian, 1968; Williams and Hallsworth, 2009). Whereas such data have formed the basis for international policy for planetary protection in relation to space exploration missions (see below), sugar‐rich substrates have very limited applicability to those extraterrestrial habitats with which we are familiar. Historically, the accepted limit for cell division of prokaryotic microbes has been 0.755 a w (for a small fraction of halophilic species at high salt concentrations, see Grant, 2004). However, both culture‐based and culture‐independent studies provide evidence for multiplication and metabolic activity of halophilic Archaea and Bacteria down to 0.611 a w , both in their natural habitats in situ and in vitro (Javor, 1984; Yakimov et al., 2014; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted).2 Whereas the vast majority of yeasts and fungi are active somewhere within the range 1 to 0.900 a w (or within a segment of this range; for examples, see Brown, 1976; Hallsworth and Magan, 1994; Kashangura et al., 2006), only ∼12 species have been observed to grow and/or germinate at < 0.700 a w (Williams and Hallsworth, 2009; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted). Here, we discuss the evidence for microbial activity in habitats at or below 0.690 a w which represent the very fringe of the functional biosphere on Earth. Low water‐activity environments are also discussed in relation to early life on Earth, the plausibility of cell division in extraterrestrial environments which contain biologically available water and a series of unanswered scientific questions.

Water activity at the fringes of the microbial biosphere The primary physical determinants of the habitable space on Earth are temperature and water activity; these parameters are also used to designate the ‘special regions’ of Mars in which microbial cell division might feasibly take place (Beaty et al., 2006; Kminek et al., 2010).3 The temperature window over which microbes are, collectively, capable of cell division (i.e. from −18°C to +122°C; Takai et al., 2008; Chin et al., 2010) spans ≤ 40% of the entire range of temperatures to which life forms on Earth can be exposed (i.e. from approximately −90°C to ≥ 250°C for some hydrothermal vents and the deep subsurface; Fig. 1A). By contrast, environmental water‐activity values range from 1 to ∼ 0, and the functional biosphere exists between 1 and ∼ 0.60 a w . Furthermore, most cellular systems of known life forms on Earth are only active within the range, or a segment of the range, 1 to 0.900 a w (Fig. 1B; Brown, 1976; Grant, 2004); for example, there is a drop‐off in the measurable activity of soil microbiota at < 0.890 a w (Manzoni et al., 2012; Moyano et al., 2013; Stevenson and Hallsworth, 2014). However, metabolic activity and cell division has been reported below 0.900 a w for a great number of xerotolerant/philic and halotolerant/philic microbes (Brown, 1976; Grant, 2004), and even below 0.755 a w for both eukaryotic and prokaryotic species (Javor, 1984; Williams and Hallsworth, 2009; Yakimov et al., 2014; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted). Of the microbes known to multiply below 0.720, the majority (unlike Xeromyces bisporus) are not obligate osmophiles that are only capable of inhabiting sugar‐rich substrates. These include halophilic prokaryotes and xerophilic fungi such as Aspergillus penicillioides and Eurotium herbariorum (Samson and van der Lustgraaf, 1978; Williams and Hallsworth, 2009; Yakimov et al., 2014; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted).4 Even for the most xerophilic microbes thus far characterized, rates of cell division typically decrease by an order of magnitude between 0.870 and 0.770 a w , and by a further order of magnitude between 0.770 and 0.670 a w (Stevenson and Hallsworth, 2014; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted). There are only reports of cell division for between 20 and 30 microbial species or communities at ≤ 0.690 a w (see Pitt and Christian, 1968; Javor, 1984; Williams and Hallsworth, 2009; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted; see also Yakimov et al., 2014). Whereas all of these species are obligately xerophilic eukaryotes or obligately halophilic prokaryotes, which have low rates of cell division – or are incapable of growth – close to a water activity of 1, the ultimate limit for multiplication of even the most resilient strains appears to be ∼0.61 a w (Pitt and Christian, 1968; Williams and Hallsworth, 2009; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted). For microbes on Earth, therefore, biotic activity spans approximately 40% of the available water‐activity range, thus emphasizing the potency of water as a determinant of the functional biosphere. The overwhelming majority of microbial systems are metabolically active somewhere within the ranges 5°C to 40°C, and 1 to 0.900 a w , which represent even smaller portions of the environmentally pertinent temperature and water‐activity ranges, i.e. only 10% in each case (Fig. 1). Of the microbial systems characterized thus far, the 20 to 30 known to be active at ≤ 0.690 a w represent the most extreme forms of life to have penetrated low water‐activity, hostile environments (Fig. 1).5 Figure 1 Open in figure viewer PowerPoint Diagrammatic representation of collective activity (compound rates cell division and metabolic activity) for microbes on Earth in relation to prevailing environmental (A) temperatures and (B) water activities. Red bars indicate the known range for cell division of microbes (−18°C to +122°C, and 1 to 0.605 a w ), and orange dotted lines indicate for (A) the established limit for cellular metabolism (33°C), and (B) the suspected limit for physiological function of DNA (down to 0.530 a w ). The blue arrow indicates the water‐activity value (0.690 a w ) below which the material of this review focuses on. Black bars indicate the range in which the overwhelming majority of microbial activity takes place, and curves represent collective biotic activity of microbes on Earth. Yellow bars indicate safety margins used for the designation of ‘special regions’ on Mars (down to −25°C and 0.500 a w ; Kminek et al., 2010) in relation to international policy on planetary protection. Horizontal orange arrows indicate zones in which cell division may take place over extended timescales (tens to thousands years) though there is a paucity of data on this topic; this zone for temperature extends considerably below −33°C because of the possibility that chaotropic substances can enhance flexibility of macromolecular systems sufficiently to reduce the temperature minima for microbial activity by a further 10°C to 20°C (Chin et al., 2010). Although the chao‐kosmotropic activity of hydrazine, which is used as spacecraft fuel to launch back to Earth, has not been quantified to date, this antimicrobial substance (Kane and Williamson, 1983) has a number of chemical and behavioral properties of chaotropic compounds (Cray et al., 2013b). In the event of a spacecraft collision, if both microbial cells and fuel were released into the Mars regolith, the hydrazine could potentially enhance macromolecular flexibility of cellular membranes, proteins, etc (Bhaganna et al., 2010). Some reports have alluded to the possibility of microbial growth and metabolism at the otherwise unprecedented water‐activity values of 0.382 (for deep‐sea halophiles in MgCl 2 ‐saturated brine; van der Wielen et al., 2005), < 0.450 (for halophiles in the CaCl 2 ‐rich, Antarctic Don Juan Pond; Siegel et al., 1979), 0.500 (Actinobacteria isolated from algal mats and cultured in soil‐based substrates; Doroshenko et al., 2005; Doroshenko et al., 2006; Zvyagintsev et al., 2009; Zvyagintsev et al., 2012), 0.570 (for halophiles in acidic, saline lakes; Mormile et al., 2009), 0.600 [for germination of Wallemia sebi (a xerophilic basidiomycete) on high‐sugar substrates; Frank and Hess, 1941] and 0.600 [reported value for optimum growth of halophiles (Jaenicke and Bohm, 1998), and biotic activity in salt lakes (Cobucci‐Ponzano et al., 2006)]. Some of these values were hypothetical (see below), and the other claims have not been accepted or have been refuted by authors of a number of subsequent studies (Pitt and Christian, 1968; Wynn‐Williams, 1996; Beaty et al., 2006; Hallsworth et al., 2007; Kminek et al., 2010; Oren, 2011; Stevenson and Hallsworth, 2014; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted).6 The Don Juan Pond (located within the McMurdo Dry Valleys, Antarctica) is a CaCl 2 ‐saturated brine pool situated in a closed basin and fed by seasonal meltwater streams and deliquescent seepages, both of which are thought to deliver CaCl 2 to the lake (Dickson et al., 2013). Its volume fluctuates but is typically ∼ 3000 m3 (slightly larger than an Olympic swimming pool), and it is among the most saline large‐scale bodies of water known on Earth. This pond rarely, if ever, freezes despite winter temperatures of ≤−51°C (Siegel et al., 1979; Marion, 1997; Grant, 2004). While annual temperatures of the pond's water and the surrounding sediments are occasionally above 0°C, they remain below −20°C for the majority of the year (Samarkin et al., 2010) and, at these temperatures, microbial cell division has not been observed (for references, see Chin et al., 2010; Kminek et al., 2010).6 Saturated solutions of divalent chloride salts, as found in the Don Juan Pond, are highly chaotropic and are therefore likely to prevent microbial growth (and may even be sterile environments; Duda et al., 2004; Duda et al., 2005; Hallsworth et al., 2007; Cray et al., 2013a; Cray et al., 2013b; Oren, 2013; Yakimov et al., 2014). Nitrous oxide emissions recorded from the surrounding sediments, frequently attributed to the biological transformation of nitrogenous compounds, are apparently the result of abiotic reactions between brine nitrates and FeII‐bearing minerals (Samarkin et al., 2010). The water activity of the MgCl 2 ‐dominated, deep‐sea hypersaline brine studied by van der Wielen and colleagues (2005) is ∼ 0.382 at the in situ temperature of 14.5°C (Winston and Bates, 1960; Hallsworth et al., 2007). Culture‐dependent and culture‐independent studies of this and comparable brines, and investigations into the biophysics of macromolecular interactions, indicate that its potent chaotropicity prohibits life processes (even at water activity values which would otherwise be permissive for cell division) (Hallsworth et al., 2007; Yakimov et al., 2014). This finding is consistent with the behaviour and hostility of solutions of comparable salts (Winston and Bates, 1960; Hallsworth et al., 2003a; Duda et al., 2004; Kminek et al., 2010; Cray et al., 2013a,b; Oren, 2013). Speculations that microbial metabolism and cell division occur at ∼ 5 M MgCl 2 are inconsistent with: (i) the microbiology of the Dead Sea that approaches a condition of sterility when MgCl 2 concentrations become elevated, but are nevertheless below 3 M (Oren, 1999; 2010; 2013), or (ii) the CaCl 2 ‐dominated Don Juan Pond (Siegel et al., 1983; Samarkin et al., 2010; Oren, 2013) where concentrations of divalent chloride salts reach critical concentrations which are prohibitive for all life processes (Hallsworth et al., 2007; Cray et al., 2013b; Oren, 2013; Yakimov et al., 2014). Although there is a theoretical possibility that some microbes have evolved specialized substances and/or structures that insulate cells from such hostile habitats while permitting biotic activity (e.g. highly kosmotropic compatible solutes; Wyatt et al., 2014a), to our knowledge, no such structures have yet been reported for any microbial species in any type of extremely chaotropic (e.g. Hallsworth et al., 2007; Yakimov et al., 2014) or low water activity (≤ 0.600) environment. Reports of germination and subsequent cell division during germ‐tube formation of several Actinobacteria [i.e. Streptomyces albidoflavus (syn. Streptomyces odorifer), Streptomyces rectiviolaceus and a Micromonospora strain] at 0.500 a w (Doroshenko et al., 2005; 2006; Zvyagintsev et al., 2009; 2012) are not consistent with data acquired by others (Stevenson and Hallsworth, 2014). Recent studies have demonstrated that none of these species was capable of growth below 0.895 a w , and the theoretical water‐activity minimum for the most xerotolerant (a strain of Streptomyces albidoflavus) is ∼ 0.877 (Stevenson and Hallsworth, 2014). Proposed limits of 0.570 or 0.600 a w for biotic activity of halophiles were speculative (i.e. not derived from determinations of water activity; Jaenicke and Bohm, 1998; Mormile et al., 2009; Cobucci‐Ponzano et al., 2006), and likely sources of experimental error in studies of W. sebi germination have been discussed previously (Pitt and Christian, 1968). Furthermore, multiplication of microbes in terrestrial brine lakes which can reach values below 0.600 a w may have actually occurred at higher water‐activity values that resulted from seasonal and weather‐related fluctuations in salt concentration (Oren, 1988; 1993; Cobucci‐Ponzano et al., 2006; Mormile et al., 2009). Although the established temperature minima for multiplication of the most psychrophilic microbes are in the region of −15°C to −18°C (Collins and Buick, 1989; Chin et al., 2010), there are numerous sources of evidence for metabolic activity considerably below this range (Fig. 1A; for references, see Kminek et al., 2010; J. D. Rummel, D. W. Beaty, M. A. Jones, C. Bakermans, N. G. Barlow, P. Boston, V. Chevrier, B. Clark, J.‐P. de Vera, R. V. Gough, J. E. Hallsworth, J. W. Head, V. J. Hipkin, T. L. Kieft, A. S. McEwen, M. T. Mellon, J. Mikucki, W. L. Nicholson, C. R. Omelon, R. Peterson, E. Roden, B. Sherwood Lollar, K. L. Tanaka, D. Viola and J. J. Wray, unpublished). By contrast, there is a paucity of data to demonstrate metabolic activity below the accepted water‐activity minimum for microbial cell division (i.e. 0.605; Fig. 1B; Kminek et al., 2010; Yakimov et al., 2014; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted).6 In relation to the water‐activity limit for life, it is noteworthy that trehalose, a hygroscopic substance (Cray et al., 2013a) that accumulates in desiccated microbial cells (e.g. Wyatt et al., 2014b) and may facilitate the acquisition and retention of water, cannot efficiently absorb water from the vapour phase at equilibrium relative humidities of much less than ∼ 50%, equivalent to 0.500 a w (Fakes et al., 2000). Some enzymes (especially some lipases) can remain catalytic below 0.500 a w , other enzymes can become highly inefficient as their hydration decreases, and others can lose their catalytic activity at water activities below the known limits for microbial multiplication (Dunn and Daniel, 2004; Kurkal et al., 2005; Lopez et al., 2010), though the implications of these findings for the physiological limits of cellular function at low water‐activity have yet to be established. There is evidence that DNA becomes disordered, and is therefore no longer transcribable, below a water activity of 0.550 (Falk et al., 1963). Furthermore, strand breaks have been recorded at 0.530 a w in bacterial cells (Asada et al., 1979).7 It has, therefore, long‐been considered unlikely that cellular systems could function at water activities substantially lower than 0.600 (Pitt, 1975; Brown, 1976; Brown, 1990; Sutton and Hildebrand, 1985; Kminek et al., 2010; Stasic et al., 2012; J. D. Rummel, D. W. Beaty, M. A. Jones, C. Bakermans, N. G. Barlow, P. Boston, V. Chevrier, B. Clark, J.‐P. de Vera, R. V. Gough, J. E. Hallsworth, J. W. Head, V. J. Hipkin, T. L. Kieft, A. S. McEwen, M. T. Mellon, J. Mikucki, W. L. Nicholson, C. R. Omelon, R. Peterson, E. Roden, B. Sherwood Lollar, K. L. Tanaka, D. Viola and J. J. Wray, unpublished). However, interactions between the various factors which determine the biophysical limits for cellular integrity and biotic activity at low water‐activity are complex and have yet to be fully elucidated. Macromolecular integrity and functionality can depend on the net effect of prevailing conditions such as temperature, chao‐/kosmotropicity, pressure and water activity (Hallsworth 1998a; Hallsworth et al., 2007; Williams and Hallsworth, 2009; Bhaganna et al., 2010; Chin et al., 2010; Yakimov et al., 2014; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted) and it may be possible that, in some as‐yet‐undiscovered environments, cells are capable of metabolism at < 0.600 a w .

Microbial cell division via utilization of water which is not in the bulk‐liquid phase Water is more or less ubiquitous on Earth and in other parts of the Solar System (Bradley et al., 2014; Küppers et al., 2014); it may be present within the atmospheres, subsurface, rocks and regolith, polar ice sheets, glaciers, and/or subsurface oceans of planetary bodies, in vapour plumes extruded into space, and – indeed – within space itself.8 Whereas here on Earth, we tend to be most familiar with water in its bulk‐liquid phase, in both terrestrial and extraterrestrial environments, it can also be present in a variety of forms. In addition to ice and vapour, these include thin, aqueous films on/at various types of surfaces and interfaces; as molecules hydrating mineral, organic and other substances (Kminek et al., 2010; Toner et al., 2014; J. D. Rummel, D. W. Beaty, M. A. Jones, C. Bakermans, N. G. Barlow, P. Boston, V. Chevrier, B. Clark, J.‐P. de Vera, R. V. Gough, J. E. Hallsworth, J. W. Head, V. J. Hipkin, T. L. Kieft, A. S. McEwen, M. T. Mellon, J. Mikucki, W. L. Nicholson, C. R. Omelon, R. Peterson, E. Roden, B. Sherwood Lollar, K. L. Tanaka, D. Viola and J. J. Wray, unpublished); and inside minerals in the form of fluid inclusions. Liquidity of water is determined by temperature, pressure, the presence of solutes and/or gases, and molecular interactions between other materials or substances and water molecules – as well as processes such as salt deliquescence, sublimation of ice, frost formation, condensation or dew formation on surfaces or within the gaseous phase, aerosol formation, and precipitation (Watanabe and Mizoguchi, 2002; Jepsen et al., 2007; Argyris et al., 2008; Möhlmann, 2008; 2009; 2012; Chin et al., 2010; Pavlov et al., 2010; Bing and Ma, 2011). Thin aqueous films can exist on various surfaces including those of ice and biological and mineral structures, and the water within these films can remain in the liquid phase under a wide range of conditions (Pearson and Derbyshire, 1974; Raviv et al., 2001; Wolfe et al., 2002; Jepsen et al., 2007; Möhlmann, 2004; Möhlmann, 2008; Möhlmann, 2009; Möhlmann, 2011; Möhlmann, 2012; J. D. Rummel, D. W. Beaty, M. A. Jones, C. Bakermans, N. G. Barlow, P. Boston, V. Chevrier, B. Clark, J.‐P. de Vera, R. V. Gough, J. E. Hallsworth, J. W. Head, V. J. Hipkin, T. L. Kieft, A. S. McEwen, M. T. Mellon, J. Mikucki, W. L. Nicholson, C. R. Omelon, R. Peterson, E. Roden, B. Sherwood Lollar, K. L. Tanaka, D. Viola and J. J. Wray, unpublished). The depth of thin films can range from > 1 mm to a monolayer of water molecules (∼ 0.3 nm; Möhlmann, 2004; Möhlmann, 2005), and they can be stable (Möhlmann, 2012) or highly ephemeral (Burkhardt and Hunsche, 2013). At the temperatures and pressures which typically prevail in Earth's biosphere, aqueous films of ∼ 1 mm are primarily made up of water which is biologically available (e.g. Qvit‐Raz et al., 2008, Burch et al., 2013). Whereas, we speculate that single‐monolayer films do not provide water that can be accessed by cellular systems. It has been suggested that microbes can utilize fluid films that have a mean thickness equivalent to that of three water molecules (Harris, 1981; Beaty et al., 2006). This hypothesis, however, may be inconsistent with the lack of solute diffusion in very thin films (Derjaguin and Churaev, 1986; Hu and Wang, 2003), which indicates that the water in films as thin as this is not in the liquid phase.9 Despite the circumstantial evidence (see also Rivkina et al., 2000), there is a paucity of data that convincingly demonstrate that water is biologically available in films of less than three water molecules deep. There are several possible sources of liquid water in otherwise desiccated and cold environments that resemble those which are characteristic of Mars, e.g.: (i) interfacial water present as a thin film (sometimes equivalent to a depth of only one or several water molecules) forming on mineral surfaces by adsorption or, on ice, as pre‐melted ice (Dash et al., 2006; Möhlmann, 2011), (ii) brines forming on salt crystals via deliquescence, (iii) as the fluid inclusions of ice and salts or other minerals, and (iv) subsurface meltwater below an ice covering due to a solid‐state ‘greenhouse effect’ (as described below) (Möhlmann, 2011). Deliquescence processes represent a particularly effective mechanism by which liquid water can be generated on Earth and, almost certainly, in extraterrestrial locations (Möhlmann, 2011). The condensing water vapour can potentially reach the dry weight of the deliquescent salt, and will exceed it if the humidity exceeds the deliquescence relative humidity. Deliquescence of NaCl, as equilibrium relative humidity increases from 65% to 80%, can be observed in Movie S1. Most salts (and, indeed, many organic substances) are hygroscopic and will attract water to their surface at equilibrium relative humidities of ≤ 100%. Each salt becomes deliquescent at a specific relative humidity, thereby dissolving as the water vapour condenses. The deliquescence relative humidity for a given salt and its (usually slight) temperature dependence quantitatively correspond to both the water‐activity values of, and equilibrium relative‐humidity values for, saturated solutions of a given salt (Winston and Bates, 1960). If the equilibrium relative humidity is higher than a salt's deliquescence relative humidity, the water activity of the salt solution will equilibrate with the relative humidity of the atmosphere, so the salt solution will become more dilute. Mixtures of substances (e.g. mixtures of different salts or salts plus sugars) will have a deliquescence relative humidity below that of each individual component (Mauer and Taylor, 2010). In addition to the reduced water activity, salts also reduce the freezing point of water, and cryobrines may be stable far below the melting point of water, e.g. under Martian conditions (Möhlmann, 2011; Martínez and Renno, 2013). Intriguingly, one recent study indicates that deliquescence of specific salts can occur, under Martian conditions, when salts are in contact with and obtaining water from ice (Fischer et al., 2014). Within the Earth's biosphere, brine formation may play a role for diverse microbial species – especially those that are halotolerant or halophilic – which are located within bioaerosols, or on mineral or biological surfaces (e.g. leaf surfaces) and are exposed to humid air (Potts, 1994). For example, adapted species can reproduce within the phyllosphere of salt‐exuding desert plants (Qvit‐Raz et al., 2008; Burch et al., 2013) and, at subzero temperatures, in supercooled water in the atmosphere (Sattler et al., 2001). Pseudomonas syringae, which is not halophilic, is a species widely transported within bioaerosols and its cells are highly effective as ice nuclei because they have protein coatings that cause water to freeze at relatively warm temperatures (Christner et al., 2008; Morris et al., 2014). Being surrounded by ice, they may benefit from water provided by the (internal) formation of thin films caused by the penetration and retention of shortwave radiation within the ice (i.e. by a solid‐state ‘greenhouse effect’). Pseudomonads (and other microbes) can produce substances such as hygroscopic biosurfactants and alginate that can attract and retain water within the vicinity of the cell (Chang et al., 2007; Burch et al., 2014). Microbes can obtain water from the vapour phase, a process that has been observed in lichens (Pintado and Sancho, 2002; Lange et al., 2006) as well as the propagules of various species (Waldham and Halvorson, 1954; Pasanen et al., 1991; Reponen et al., 1996; Bekker et al., 2012). Other studies have demonstrated that microbial cells also generate considerable quantities of water via their metabolic activity (Oriol et al., 1988; Nagel et al., 2001; Marcano et al., 2002; Kreuzer‐Martin et al., 2005; 2006; de Goffau et al., 2011), up to 70% of the cell's water according to radio‐labelled gas uptake experiments (Kreuzer‐Martin et al., 2005; 2006). Spore germination of powdery mildews, such as by the Erysiphe and Uncinula species, has been observed, at low equilibrium relative humidities (0% to 10%) without a visible extracellular source of liquid water (Brodie and Neufeld, 1942; Manners and Hossain, 1963; Carroll and Wilcox, 2003), although it is not clear whether condensation processes and/or thin films might act to shuttle water to the cell. Desiccated lichens are able to absorb water at an equilibrium relative humidity of ≥ 82% and thereby commence photosynthesis (Pintado and Sancho, 2002; Lange et al., 2006). Various lines of evidence suggest that microorganisms may be capable of cell division without an extracellular supply of liquid water (see also Miller and Chibnall, 1932; Yarwood, 1950; Peterson and Cowling, 1973; Lange et al., 1986; Lange et al., 1994; J. D. Rummel, D. W. Beaty, M. A. Jones, C. Bakermans, N. G. Barlow, P. Boston, V. Chevrier, B. Clark, J.‐P. de Vera, R. V. Gough, J. E. Hallsworth, J. W. Head, V. J. Hipkin, T. L. Kieft, A. S. McEwen, M. T. Mellon, J. Mikucki, W. L. Nicholson, C. R. Omelon, R. Peterson, E. Roden, B. Sherwood Lollar, K. L. Tanaka, D. Viola and J. J. Wray, unpublished). However, there is a paucity of convincing data to irrefutably affirm this hypothesis. Furthermore, systematic studies of water‐activity limits for cell division of phylogenetically diverse extremotolerant and extremophilic microbes10 suggest that cell division would be implausible at values much below 0.600 a w (i.e. 60% equilibrium relative humidity) (Pitt and Christian, 1968; Brown, 1976; Williams and Hallsworth, 2009; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted). This question is equally pertinent to life on Earth, and the aqueous milieux found elsewhere in the Solar System (not least in relation to planetary protection).

Implications for the evolution of microbial life on Earth The most solute‐tolerant Bacteria and Archaea are only able to grow at their water‐activity minima under hypersaline conditions (i.e. extreme, obligate halophiles).11 Some of these organisms thrive under conditions that would have been available in saline environments on the early Earth. Extremely halophilic Archaea and Bacteria typically exhibit higher optimal growth temperatures than those of mesophilic or moderately halophilic comparators (Ratkowsky et al., 1983; Oren, 1992; Ramos‐Cormenzana, 1992; Robinson et al., 2005). Indeed, the minimum and maximum NaCl concentrations at which growth of extreme halophiles can occur increase at higher temperatures (Mullakhanbhai and Larsen, 1975; Vreeland and Martin, 1980; Quesada et al., 1987; Rodriguez‐Valera, 1992). There is some debate regarding the temperature of the early seas (those of ∼ 3.5 billion years ago); earlier estimates of 70–80°C (Knauth and Lowe, 2003) are now considered to be too high (the δ18O values on which the calculations were based may have been skewed due to inputs of hydrothermal fluids). More recent estimates based on analysis of oxygen and hydrogen isotopes (i.e. δ18O and δD respectively) are about 40°C (Blake et al., 2010). However, the high levels of heat flow within the mantle on the early Earth drove a highly active hydrothermal circulatory system that contributed hot, salty (de Ronde et al., 1997), silica‐rich fluids to the local environment (Westall, 2012). It has been proposed that primordial life may have first occurred within hypersaline environments on early Earth (Dundas, 1998), and recent evidence suggests that the abiotic formation of primitive versions of extant proteins can indeed occur in the presence of NaCl (Longo et al., 2013; Longo and Blaber, 2014). Understanding the way in which water‐condensing chemical reactions could have led to the emergence of key biomolecules (e.g. peptides and nucleic acids) is essential to understanding the origins of life (Da Silva and Holm 2014, and references therein). Prokaryote life (anaerobic) was relatively abundant in these early environments and left behind numerous signatures of its presence (Westall, 2012). There are stratified salt deposits of various ages throughout large regions of the Earth, indicating that concentrated salt‐waters/brines have existed across the planet's geologic history (Warren, 2010). Direct association of an early photosynthetic microbial community with evaporitic conditions is documented in 3.33‐billion‐year‐old volcanic sands from the Barberton greenstone belt, South Africa (Fig. 2; Westall et al., 2001; 2006; 2011). The uppermost layers of a desiccated biofilm, formed on sediments deposited in shallow waters that were partially exposed to air, are interlayered with tiny evaporate crystals (microns in size and including aragonite, gypsum, halite and magnesium calcite; Fig. 2). Evaporitic precipitates have been described from other formations on the early Earth, including the 3.42‐billion‐year‐old Buck Reef Chert in Barberton (Lowe and Fisher‐Worrell, 1999) and the 3.43‐billion‐year‐old Strelley Pool Chert of the Pilbara in Australia (Allwood et al., 2007). The early phototrophs were quite advanced on the evolutionary scale compared with chemotrophs. Although, to date, no direct association of chemotrophic biosignatures with the early evaporitic deposits has been identified, these more primitive organisms were nevertheless also common (Westall, 2012; Westall et al., 2013). Experiments simulating the entry of meteorites containing microorganisms into the Earth's atmosphere have shown that, if primitive cells did reach the early Earth through panspermia: (i) phototrophs could not have been transported to Earth by these means (Cockell et al., 2007), and (ii) if resilient forms of life were hidden in meteorites, they would need to be buried at depths of at least 5 cm in cracks within the meteorite in order to withstand the heat of entry (Foucher et al., 2010). Figure 2 Open in figure viewer PowerPoint Early Archaean microbes and evaporites; example from the 3.33‐billion‐year‐old Josefsdal Chert, Barberton Greenstone Belt: (A) layer of evaporite minerals interbedded with layers of a photosynthetic microbial biofilm, (em) evaporite minerals, and (B) details of the diversity of minerals encrusted on the surface of the biofilm. They include here pseudomorphs (silica replaced) of acicular aragonite and lozenge‐shaped gypsum. Reproduced from Westall and colleagues (2006) with permission from The Royal Society Press. Regardless of how (and where) life originated, it seems most likely that it was prokaryotes (known to have preceded eukaryotes by ∼ 2 billion years) in hypersaline environments which were first able to multiply close to 0.605 a w , as documented by the 3.33 Ga‐old, evaporite‐coated, anoxygenic photosynthetic microbial biofilm noted above (Westall et al., 2006; 2011). In relatively unperturbed, sediment‐starved environments, these photosynthetic films built up into three dimensional, dome‐shaped stromatolites (e.g. Allwood et al., 2007). Intriguingly, molecular analysis of modern stromatolite communities revealed that 74% of the Archaea present were closely related to the Halobacteria (Burns et al., 2004), which frequently dominate hypersaline environments (Oren, 2002; Cray et al., 2013b; Oren and Hallsworth, 2014). These prokaryotic halophiles were exposed to, and presumably inhabited, evaporitic environments containing elevated concentrations of magnesium and characterized by water activities of considerably less than 0.755 (and can, indeed, be considerably below 0.600 a w , depending on salt concentrations; Winston and Bates, 1960; Hallsworth et al., 2007; Yakimov et al., 2014; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted). Indeed, the signatures of past life forms, including stromatolites, can be common in evaporitic deposits (Rothschild and Mancinelli, 2001). Much later, and presumably in land‐based (rather than marine) habitats, the Eukarya must have developed a similar resilience during growth at high concentrations of solutes which are produced via biogenic activity, namely sugars and polyols. Indeed, the most halophilic Eukarya are considerably less salt tolerant than their bacterial and archaeal counterparts, and it may be that the prokaryotes are yet to evolve an ability to grow at low water‐activity in non‐saline substrates (their current record is in the range 0.850 to 0.800; Lievens et al., 2014; R. Santos, A. Stevenson, C. C. C. R. de Carvalho, I. R. Grant, I. R. and J. E. Hallsworth, submitted; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted).

Extraterrestrial, aqueous milieux which resemble fertile habitats on Earth Liquid water was, and is still, present in numerous locations in the Solar System. On Mars, for example, there is abundant geomorphological evidence for the presence of liquid water on the planet in the past (Carr, 2006) and possibly even, ephemerally, in the present (Möhlmann, 2011; McEwen et al., 2014; J. D. Rummel, D. W. Beaty, M. A. Jones, C. Bakermans, N. G. Barlow, P. Boston, V. Chevrier, B. Clark, J.‐P. de Vera, R. V. Gough, J. E. Hallsworth, J. W. Head, V. J. Hipkin, T. L. Kieft, A. S. McEwen, M. T. Mellon, J. Mikucki, W. L. Nicholson, C. R. Omelon, R. Peterson, E. Roden, B. Sherwood Lollar, K. L. Tanaka, D. Viola and J. J. Wray, unpublished). Such evidence includes the formation of secondary minerals through the aqueous alteration of the basaltic rocks that cover the surface of the planet (e.g. Carter et al., 2013; Martínez and Renno, 2013). It has been calculated that the water activities of evaporite deposits and bodies of saline water on early Mars were as high as 0.780 to 0.860 (Tosca et al., 2008), which is well within the ranges for microbial species from each domain of life (Javor, 1984; Grant, 2004; Williams and Hallsworth, 2009; Stevenson and Hallsworth, 2014; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted). There is evidence for various brines on Jupiter's moon Europa (Fig. 3A) that are composed primarily of water and salts such as MgSO 4 , Na 2 SO 4 and/or Na 2 CO 3 (and, in some cases, also contain sulfuric acid; Muñoz‐Iglesias et al., 2013). Saturated solutions of these salts have water‐activity values of 0.900, 0.930 and 0.920 respectively (at 20°C, 1 atm; Winston and Bates, 1960), although it is currently unclear what the values would be under the prevailing conditions on Europa. At the lower temperatures, and the in situ pressures, on Europa, the solubility of ions and, conversely, the precipitation of salts can also vary leading to increases in water activity (Marion et al., 2003; 2005); the water activity of a saturated Na 2 CO 3 solution at 10°C, for example, is 0.990 (Winston and Bates, 1960). Whereas water‐activity values for individual brines will vary according to their ionic composition (and pH, which also influences solubilities of some salts), it seems likely that the in‐situ water activities span the entire range for known life (Javor, 1984; Williams and Hallsworth, 2009; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted). Figure 3 Open in figure viewer PowerPoint Views of two planetary moons which are known to have an abundance of water, some of which may be present as subsurface oceans: (A) the icy surface of Europa, and (B) jets composed of water vapour, ice particles and organic compounds released from beneath the surface of Enceladus. Courtesy NASA/JPL‐Caltech. Water has also been identified in asteroidal materials, for example the Monahans (1998) H5 chondrite which contained hypersaline fluid inclusions composed predominantly of saturated NaCl (Zolensky et al., 1999) having a water activity of 0.760 at 20°C and 0.750 at 2°C at 1 atm (Winston and Bates, 1960), although these values will vary with pressure. Fluid inclusions have been identified in an increasing number of asteroidal specimens including the Zag (1998) meteorite (Rubin et al., 2002). Furthermore, organic molecules have been detected in the fluid inclusions of some of these asteroidal bodies (e.g. Fries et al., 2012); thus, the composition of these fluids can be close to those of the media and substrates in which halophiles occur. For instance, halophiles in hypersaline fluid inclusions of salt crystals from evaporite deposits contain Archaea, Bacteria and algae (Dunaliella species).12 Many NaCl‐saturated habitats contain a remarkably high microbial biomass and are characterized by intense competition (Antón et al., 2002; Daffonchio et al., 2006; Baati et al., 2008; Elevi Bardavid et al., 2008; Khemakhem et al., 2010) during which some species – which are known as ‘microbial weeds’ (Cray et al., 2013a; Oren and Hallsworth, 2014) – achieve dominance of the communities including Archaea, Bacteria and Eukarya (e.g. Haloquadratum walsbyi, Salinibacter ruber and Dunaliella salina; for references, see Cray et al., 2013a; Oren and Hallsworth, 2014). The microbes that dominate and/or are most frequently isolated from the fluid inclusions of salt crystals found in evaporite deposits include a number of species known to be capable of cell division in the range 0.739 to 0.611 (or their close relations, such as Dunaliella, Halocarcula, Halobacterium, Halococcus, Halorubrum and Natrinema spp.: McGenity et al., 2000; Stan‐Lotter et al., 2000; Schubert et al., 2009b; Lowenstein et al., 2011; Gramain et al., 2011; A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted). In relation to water activity, the biotic activity of microorganisms – including halophiles – is plausible for some of the aqueous milieux found in extraterrestrial environments. Indeed, some of these locations resemble highly fertile habitats for known halophiles (see also A. Stevenson, J. A. Cray, J. P. Williams, R. Santos, R. Sahay, N. Neuenkirchen, C. D. McClure, I. R. Grant, J. D. R. Houghton, J. P. Quinn, D. J., Timson, S. V. Patil, R. S. Singhal, J. B. Antón, J. Dijksterhuis, A. D. Hocking, B. Lievens, D. E. N. Rangel, M. A. Voytek, N. Gunde‐Cimerman, A. Oren, K. N. Timmis, T. J. McGenity, and J. E. Hallsworth, submitted). Planets which are neither too close to nor too far from a star and could, theoretically at least, accommodate active biological systems are said to be in the circumstellar habitable zone or Goldilocks zone of their respective solar system (Strughold, 1953). This designation is based on criteria, such as size of the planet and its absolute distance from the star it orbits, whether luminosity could permit photosynthesis, having surface temperatures which are biologically permissive for at least some of the time (variously defined as 0°C to 100°C, or −25°C to +122°C; Franck et al., 2007; Takai et al., 2008; Kminek et al., 2010; Harrison et al., 2013), and/or whether they have liquid water (Rampino and Caldeira, 1994; Von Bloh et al., 2011). However, these criteria (and indeed the habitable‐zone concept) have limited applicability or validity for a variety of reasons. Ecosystems exist on Earth which do not depend on photosynthetic activity (Chivian et al., 2008; Teixeira et al., 2013) and, indeed, the earliest forms of life were not photosynthetic (Westall, 2012); furthermore, there is circumstantial evidence that an extracellular source of liquid water is not obligatory for microbial life (see above). What is more, biologically permissive conditions may prevail in specific environments or substrates on otherwise hostile planetary bodies (for example in relation to moons of Saturn, see Raulin, 2008; Nimmo et al., 2007; Parkinson et al., 2008). And finally, various activities of solutes can both prevent freezing of water and expand biotic windows of microbes, and may be able to do so to a greater degree than has yet been recorded (see below; Chin et al., 2010; J. D. Rummel, D. W. Beaty, M. A. Jones, C. Bakermans, N. G. Barlow, P. Boston, V. Chevrier, B. Clark, J.‐P. de Vera, R. V. Gough, J. E. Hallsworth, J. W. Head, V. J. Hipkin, T. L. Kieft, A. S. McEwen, M. T. Mellon, J. Mikucki, W. L. Nicholson, C. R. Omelon, R. Peterson, E. Roden, B. Sherwood Lollar, K. L. Tanaka, D. Viola and J. J. Wray, unpublished). Water can remain liquid at temperatures far lower than those known to permit microbial cell division (i.e. approximately −18°C; see references in Chin et al., 2010; J. D. Rummel, D. W. Beaty, M. A. Jones, C. Bakermans, N. G. Barlow, P. Boston, V. Chevrier, B. Clark, J.‐P. de Vera, R. V. Gough, J. E. Hallsworth, J. W. Head, V. J. Hipkin, T. L. Kieft, A. S. McEwen, M. T. Mellon, J. Mikucki, W. L. Nicholson, C. R. Omelon, R. Peterson, E. Roden, B. Sherwood Lollar, K. L. Tanaka, D. Viola and J. J. Wray, unpublished). Liquid water (in various forms, from thin films to underground oceans) may be found in many environments on Mars as well as planetary moons (Europa, Ganymede, Enceladus, etc.). Diverse lines of evidence suggest that both photosynthetic and non‐photosynthetic microbes may be capable of metabolism and cell division by hygroscopic absorption of water vapour and/or acquiring water from their substratum (as a sole extracellular source of water) both in vitro and in their natural habitats on Earth,13 and utilize a variety of mechanisms for the acquisition and retention of water (e.g. production and accumulation of trehalose and other hygroscopic substances which optimize the acquisition and retention of water, morphological changes which minimize water loss, hydrotactic responses, inhabiting high humidity niches, and construction of soil features to enhance water capture and retention; Garcia‐Pichel and Pringault, 2001; Garvie et al., 2008; de Goffau et al., 2011; Williams et al., 2012; Rajeev et al., 2013; Zakharova et al., 2013). Furthermore, as noted above, some microbial cells can generate vast quantities of water via their metabolic activities (Miller and Chibnall, 1932; Peterson and Cowling, 1973; Oriol et al., 1988; Nagel et al., 2001; Marcano et al., 2002; Hocking, 2003; Kreuzer‐Martin et al., 2005; 2006). As mentioned above, bacterial cells demonstrate that up to 70% of intracellular water may be obtained in this way, and other studies suggest that bacterial cells may be able to maintain higher intracellular water‐activity than that of the environment (de Goffau et al., 2011). The rarefied atmosphere of Saturn's moon Enceladus can contain ≥90% water vapour (Waite et al., 2006) and, whereas its surface is approximately −200°C (Brown et al., 2006), plumes of water vapour and ice which are released into space (Fig. 3B) are thought to originate in subsurface oceans that have temperatures in the range −23°C to −3°C (Nimmo et al., 2007; Parkinson et al., 2008); i.e. temperatures which are permissive for the metabolic activity of psychrotolerant and psychrophilic microbes (Collins and Buick, 1989; Chin et al., 2010; Kminek et al., 2010; Mykytczuk et al., 2013). Various salts, nitrogenous compounds and organic substances have been identified in the atmosphere of Enceladus and E‐ring ice grains of Saturn (which may originate from Enceladus) including NaCl, NaHCO 3 , NaCO 3 , N 2 , ammonia, hydrogen cyanide, CO and CO 2 , methane, acetylene and propane (Matson et al., 2007; Postberg et al., 2009; 2011). Under conditions prevalent on Earth, bioaerosols can be fertile habitats characterized by high levels of microbial diversity, biomass and metabolic activity (Fahlgren et al., 2010; Womack et al., 2010; 2012). In relation to the atmosphere of Enceladus and/or the watery plumes which it emits into space, it is intriguing to speculate what the water activity of liquid droplets in, or the humidity of, the gaseous phase (presumably close to 100%) might be and whether the temperatures within these plumes can ever be considerably higher than −200°C. It should be noted that, whereas definitive evidence from culture‐based studies of microbial systems on Earth indicate limits for cell division of approximately +122°C or −18°C (Collins and Buick, 1989; Takai et al., 2008; Chin et al., 2010; Harrison et al., 2013), circumstantial evidence from other biochemical or geochemical data suggest biotic activity under more extreme conditions (down to about −40°C, and up to approximately +140°C; Kminek et al., 2010; J. D. Rummel, D. W. Beaty, M. A. Jones, C. Bakermans, N. G. Barlow, P. Boston, V. Chevrier, B. Clark, J.‐P. de Vera, R. V. Gough, J. E. Hallsworth, J. W. Head, V. J. Hipkin, T. L. Kieft, A. S. McEwen, M. T. Mellon, J. Mikucki, W. L. Nicholson, C. R. Omelon, R. Peterson, E. Roden, B. Sherwood Lollar, K. L. Tanaka, D. Viola and J. J. Wray, unpublished). Although the Earth is located within the region allocated as the Goldilocks zone of our own solar system, it hosts many environments which do not permit life‐processes and are therefore essentially sterile due to, for example, low water‐activity, high chaotropicity, excessively high or low temperatures, pH of > 12, plus combinations of conditions such as high salt and low pH or high temperature and high pH (e.g. Brown, 1990; Hallsworth 1998a; Grant, 2004; Hallsworth et al., 2007; Harrison et al., 2013; Yakimov et al., 2014). Under all these conditions, cells also need adequate energy sources and nutrients for maintenance and growth which may require electron donors and acceptors for respiration etc. Some combinations of conditions can slightly extend extremes for growth, such as high pressure and temperatures; furthermore, survival can occur under conditions where growth cannot.14 Conversely, planetary bodies which are basically hostile to life may nevertheless harbour small‐scale, biologically permissive domains (Kminek et al., 2010; J. D. Rummel, D. W. Beaty, M. A. Jones, C. Bakermans, N. G. Barlow, P. Boston, V. Chevrier, B. Clark, J.‐P. de Vera, R. V. Gough, J. E. Hallsworth, J. W. Head, V. J. Hipkin, T. L. Kieft, A. S. McEwen, M. T. Mellon, J. Mikucki, W. L. Nicholson, C. R. Omelon, R. Peterson, E. Roden, B. Sherwood Lollar, K. L. Tanaka, D. Viola and J. J. Wray, unpublished). Solute activities represent one of the determinants for potential habitability on Earth; for example, chaotropicity can enable cellular function at low temperatures and kosmotropicity may enable cellular function in high‐temperature environments or those dominated by chaotropic substances.15 The ways in which water activity and other solute activities can interact to determine the physicochemical limits for life (e.g. Williams and Hallsworth, 2009; Chin et al., 2010) have yet to be fully characterized. Furthermore, there is little information on the way in which availability of nutrients and other resources can determine tolerance limits to physicochemical stress parameters (e.g. Daffonchio et al., 2006; J. P. Harrison, J. E. Hallsworth, and C. S. Cockell, submitted). Once the interactions between such factors are better understood, the currently accepted criteria for habitability will require revision (Beaty et al., 2006; Marion et al., 2003; Marion and Kargel, 2008; Tosca et al., 2008; Kminek et al., 2010; Harrison et al., 2013; J.D. Rummel et al., unpublished).