Wooden owl that redefines Earth's biosphere may yet catapult a fungus into space

Cellular systems are vulnerable to environmental parameters that impose thermodynamic constraints on metabolic function. These include biophysical challenges and events relating to low water-availability, solute-induced stresses, dehydration-rehydration cycles and extremes of temperature, and these can sometimes be lethal. The concept of a water-availability limit for Earth's biosphere might evoke the Sahara, Atacama and other deserts where water is scarce, or subzero environments where any water is locked up as ice. However, if a cell is dried then, whereas it may remain viable, life processes cease, from an existential viewpoint it is debatable whether life exists/occurs under such conditions at all. Regardless of this, water stress also occurs in microbial habitats that are wet, even within bodies of water. Indeed, it is arguably implausible that any microbial system can ever function in a stress-free mode, and this can often be due oxidative stress caused by reactive oxygen species as well as oscillating suboptimal or supraoptimal water activity (Hallsworth, 2018). Water activity, that is the mole fraction of water molecules in a solution, is a potent stress parameter that is numerically equivalent to relative humidity but divided by 100 (Scott, 1957). Cells respond to low water activity, osmotic stress and other water- and solute-induced stresses using a variety of mechanisms, including production of protein-stabilization proteins, increased generation of cell-available energy, adaptations of plasma-membrane composition and accumulation of soluble stress-metabolites (compatible solutes) that can act in osmotic adjustment and protect macromolecular structures, depending on the microbe and stress(es) experienced. Some microbes, known as xerophiles, are able to function optimally at much lower values of water activity than most other taxa. Here, ‘xerophiles’ refers to fungi that are capable of growth at low water activity on high-sugar or high-glycerol media (although some xerophilic fungi are also halophilic). Research into this group was historically driven by the food industry, with studies focusing on fungi that spoil high-sugar foods (Gane, 1950; Scott, 1957; Pitt, 1975; Dagnas and Membré, 2013; Biango-Daniels and Hodge, 2018). In recent years, an increasing amount of information has come to light about fungi in high-salt habitats, such as solar salterns (Gunde-Cimerman and Zalar, 2014; Nazareth and Gonsalves, 2014). Although some xerophiles are known to cause indoor contamination of building materials (Fig. 1A) and the formation of brown spots on paper (caused by the Maillard reaction and known as ‘foxing’: Arai, 2000; Piñar et al., 2015), the surfaces of domestic furniture and artefacts have generally been overlooked as a potential source of extremely xerotolerant microbes. Water availability is a potent determinant for life on Earth, and it is a quirk of history that water activity was designated a maximum value of 1. The parameter is derived using Raoult's Law (Scott, 1957), so we designate water activity in decimal fractions. For 50 years, the water-activity value at which the most-resilient microbe was thought to become non-functional is 0.605 (Pitt and Christian, 1968); equivalent to a relative humidity of 60.5%. In other words, whereas the biophysical window for microbial life on Earth spans a temperature range of approximately 160°C, the entire water-activity window for life is allocated a value of only 0.4 units (Stevenson et al., 2015a). Given that living systems are as sensitive to water stress as they are to temperature, this makes it is easy to overlook the profound impact of water activity on cellular systems. Optimal rates of growth/metabolism, and/or health, occur at different water-activity values, according mainly to the biological system. In humans and most other mammals, this is approximately 0.995 (Pirt and Thackery, 1964; Persons et al., 1987), for most fungi approximately 0.990 and for some extreme xerophiles 0.900–810 (Stevenson et al., 2015b; 2017a). In their study, Pitt and Christian isolated fungi from spoiled fruits, and determined the water-activity minima at which they were able to germinate. Aspergillus, Chrysosporium, Penicillium and other genera were present, but the most xerophilic was a Xeromyces bisporus strain (FRR 0025) that germinated at 0.605 water activity after a four-month incubation. For organismal systems, cellular water-activity tends to be closely regulated, although some plant systems and some invertebrates are able to undergo partial desiccation via anhydrobosis. Many fungi can survive dehydration but for mammals, such as ourselves, relatively small fluctuations of cellular water-activity are lethal. In relation to X. bisporus, 0.605 has been used as the seminal value upon which methodologies to preserve foods, museum specimens, artworks, documents and books have been based. In addition, the COSPAR Planetary Protection Policy, recognized by the United Nations, is based this value. This policy minimizes the risk of contamination events on extraterrestrial bodies with terrestrial microbes during space-exploration missions (Kminek et al., 2010; Rummel et al., 2014). Whereas X. bisporus has held its position as the most-extreme xerophile for five decades, there was little systematic research carried out during this time to find microbes that might be more xerophilic. It seemed difficult, therefore, to accept that strain FRR 0025, which originated from a prune, might not one day be dislodged from its number-one position. So, I set about trying to coax various microbes to multiply below this value. Optimistically, and with some naivety, we expected that achieving this would take several months. It transpired, however, that this scientific journey which began in 2001 threaded its way through several generations of research students and the three domains of microbial life; involved 126 collaborators/coauthors (from 22 countries), and sampling campaigns on two different continents; and spanned a period of almost 20 years. Working as a PhD student with Naresh Magan (Cranfield University, England, UK; 1991–1994), we exploited the phenotypic plasticity of entomopathogenic fungi to enhance intracellular accumulation of the compatible solutes trehalose, mannitol, arabitol, erythritol and glycerol. We found that the lowest molecular-weight polyols, especially glycerol, increased the vigour of, and reduced the water-activity minimum for, fungal germination and growth in vitro, and on the insect host (Hallsworth and Magan, 1994; 1995). After this, I took several postdoctoral positions, at Heriot-Watt University (Scotland, UK), Kumamoto Institute of Technology (Japan), Stellenbosch University (South Africa) and then – in the group of Kenneth N. Timmis – at University of Essex (England). The microbial model system(s) and their applications differed in each location, but the underlying theme was consistent; to identify cellular stress mechanisms and responses. This included stresses imposed by low-temperature and low water-activity, and that imposed by ethanol, urea and other chaotropic substances; that is those that entropically disordering macromolecular systems (i.e. those that are chaotropic). Whereas chaotropicity and water activity are mechanistically distinct stress parameters, the chaotrope-stress work impacted our search for microbes that can tolerate low water-activity. During the time in Essex, I worked on several projects, including the EU-funded Biotechnologies from the Deep (BIODEEP; 2001–2004), alongside halophile expert Terry J. McGenity. BIODEEP focused on the microbial ecology of the Mediterranean deep-sea anoxic brine lake Discovery that lies 3.58 km below the ocean surface. The brine of the lake body is saturated with MgCl2, and the lake's interface with the overlying seawater is a 1.5-m deep halocline with a Mg2+ gradient from 50 mM to 5.05 M (Hallsworth et al., 2007). Our study was carried out in collaboration with Michail M. Yakimov (Institute for Coastal Marine Environment, Italy), Peter N. Golyshin (Helmholtz Centre for Infection Research, Germany) and others, and the main scientific finding was that the chaotropicity of MgCl2 constrained life to the upper layers of the interface. There was no metabolic activity in evidence in the lower interface at MgCl2 concentrations of > 2.3 M. Furthermore, disparate lines of evidence indicated that the presence of kosmotropic (macromolecule-stabilizing) salts would enable microbial activity at slightly higher MgCl2 concentrations, by mitigating against the chaotropicity of the latter; a phenomenon that was confirmed in a number of other studies (Williams and Hallsworth, 2009; de Lima Alves et al., 2015; Cray et al., 2015; Yakimov et al., 2015). We also worked on NaCl-saturated deep-sea systems (Daffonchio et al., 2006), and noticed that some halophilic bacteria and Archaea exhibit very high growth rates – and even optimal growth – at NaCl saturation (0.755 water activity). However, proliferation has not been observed at lower water activity due to the finite solubility of NaCl. During the Lake Discovery study, we also worked on synthetic, MgCl2-dominated substrates and found that no halophiles can retain function in such brines even at 0.755 water activity (Hallsworth et al., 2007). So, we set about looking into how we might manufacture brines with a water-activity of < 0.755, yet a sufficiently low chaotropic activity to permit halophile metabolism. At the time, one of our colleagues commented that this kind of work ‘seems a bit like breaking records’. This was an amiable colleague, and the comment was meant to be helpful, but we didn't share that view. For me, working with life at the point where the cellular system interfaces with extreme, thermodynamic constraints is in itself motivating, and even fascinating. Understanding system failure at the biophysical limit can potentially elucidate aspects of ecophysiology even for non-extremophilic microbes. We also believed that water plays a key role under the extreme (solute-induced) conditions where even the most-resilient microbes fail (Hallsworth et al., 2003; Stevenson et al., 2015a; Ball, 2017). We recruited MSc student Richa Sahay (2003–2004) for the study of halophiles at low water-activity. Richa cultured some of the most halophilic microbes known, and determined ability to grow in synthetic brines that contained NaCl+MgCl2 and NaCl+MgCl2 + either glycerol or ethylene glycol (and some nutrients). In this way, we found that some strains can indeed proliferate below 0.755 water activity, and even down to 0.681. Analyses of data from published sources (e.g. Javor, 1984; Yoshida et al., 1991; Antón et al., 2000; Bolhuis et al., 2004; Deole et al., 2013) were later carried out by PhD student Andrew Stevenson from 2013 to 2016, who worked with me once based in Belfast. To ensure that our study was as comprehensive as possible, we asked a number of experts to join us: Ailsa D. Hocking (fungal xerophiles; CSIRO Food and Nutrition, Australia), Nina Gunde-Cimerman (fungal halophiles; University of Ljubljana, Slovenia) Josefa Antón and Aharon Oren (halophilic bacteria and Archaea; University of Alicante, Spain and The Hebrew University of Jerusalem, Israel respectively) and others. Andy's analyses revealed that some prokaryotes function at even lower water-activity values; that is down to 0.635 (Fig. 1B). Collectively, the findings showed that the respective limits for the three domains-of-life actually converge close to a water-activity value (Stevenson et al., carried out by student J. D. et al., confirmed that NaCl-saturated environments are than extreme habitats for that is that microbial can function at < 0.755, saturated NaCl not a water-activity to the microbial biosphere et al., 2018). Cray student from to involved in our water-activity studies through analyses of deep-sea brines et al., 2015). The scientific journey also its way through the stress of et al., and the of high-sugar et al., 2015). We also the evidence for based on the of the most-extreme xerophiles and halophiles at < water activity (Stevenson et al., 2015a). Whereas there is an research working on halophiles on the for the five at fungal xerophiles are only 22 The relatively low of in xerophiles however, the applications of fungal xerophiles, and the they into of Some of our other studies at the time of halophile study were focused on fungi and a de Lima at University of Essex to work on this studies of in indicated that glycerol was the most that can be used to the water activity of media Lima Alves et al., 2015). also revealed that glycerol an osmotic only and then the At the time as Richa and were working on solute-induced stresses, I a to a and two according to the been in (Fig. This to the it out impacted our research I began up a research group in University from and recruited PhD student at this time is an with deep in cellular and for the So, we were able to on the search for microbial proliferation below 0.605 water-activity. We xerophile strains by the for extreme xerophiles and these from and via sampling campaigns in three and relatively of and We began sampling by the surfaces of and surfaces of furniture and domestic some of these in The we in also included the X. bisporus strain (FRR that was isolated by Pitt and used in the study by Pitt and Christian For xerophile began by ability to grow at low water-activity using ethanol, ethylene glycerol, MgCl2, and as and that the of strains at their lowest water-activity on xerophile growth rates a of conditions activity, temperature, and the each capable of growth at water These included and from the and (from the of an from the FRR (from dried (from an in FRR (from a Xeromyces bisporus FRR (from a FRR (from and FRR (from a (Williams and Hallsworth, strains isolated during our sampling campaigns and were using a of and strains and and strains and were isolated from (Fig. that been in the which is close to the of and on and with a (and or It may be that such habitats for xerophile In to the water-activity minimum for xerophiles a range of media with glycerol or glycerol kosmotropic solutes (e.g. These studies some strains capable of growth at lower water-activity values that been (i.e. in the range (Williams and Hallsworth, 2009; Stevenson et al., These at water activity, and X. bisporus strains FRR and FRR (Stevenson et al., at strains and FRR and at X. bisporus strains FRR (Williams and Hallsworth, We were by the finding that impacted water-activity minima for xerophile but at the time not to have observed activity at The finding that X. bisporus can at 0.605 not been or during studies of fungal Furthermore, whereas of growth a minimum for some halophiles of down to there are no of activity of halophilic bacteria or Archaea at 0.635 (Stevenson et al., studies have high of microbial and growth at low water-activity growth of at et al., et al., germination of the fungal xerophile at and and data in the deep-sea brine lake at and to be evidence of and such as and et al., 2018). data have not been and have either been (Pitt and Christian, Stevenson and Hallsworth, or data any cellular or activity et al., M. M. Yakimov et al., in We carried out analyses of growth for X. bisporus and strains with the xerotolerant and found that the fungal cell is sensitive to of water activity of at (Stevenson et al., 2015a). We the for water-activity based on to be to an equivalent of (Hallsworth and Stevenson et al., on a might water from and and may also water via cellular metabolism. that the mole fraction of water in and the are in (i.e. water activity and relative humidity are not the fungal cell would water from the with the but little work has been on this to the which I was a was et al., and we whether microbes proliferate by water from the The that microbes may be able to grow any source of water was a to some on and at our by Institute of and was the of that can on surfaces So, microbes on surfaces not known to may potentially a of water via formation that is to the This me to a with of a out studies into on plant We used a of formation on a plant to how can act as sources of water for (Stevenson et al., 2015a). are known to water from the and to water through metabolic but whether or not an source of water is for microbial proliferation Stevenson was in the of PhD work at we to have a to the water-activity limit for via studies of germination on a range of media with glycerol or glycerol other number of xerophiles were for this X. bisporus strains FRR 0025, FRR (from spoiled FRR and FRR strain FRR strain FRR (from strain FRR (from strain FRR (from spoiled from the CSIRO Food and and strains and and strain (Stevenson et al., that contained high of intracellular glycerol were from on were in of glycerol, or glycerol other solutes and these were used to the range of low water-activity this high-glycerol study system to of germination vigour at low water-activity, and reduced the water-activity minima for germination of to our however, germination not occur at X. bisporus germinated down to to and to water So, once our to microbial activity at 0.605 been indicated a water-activity minimum for germination of X. bisporus and of colleagues to that are and that there may be no activity in below I the such but also that these extreme xerophiles were capable of more than we observed within our system. In the of PhD by this time, I with to one Many xerotolerant species were from our sampling including from the of a dried in from an Penicillium and from a in from the of a dried of in and and from (Fig. in (Williams and Hallsworth, 2009; The most xerophilic however, from the surfaces of (Fig. (Williams and Hallsworth, studies that were carried out to parameters such as temperature and that strain may be the most xerophilic strain in our (Fig. and it germinated and growth at (Stevenson et al., 2015b; Furthermore, has an and and is a that is (Stevenson et al., and J. Hallsworth, in more xerophilic than X. bisporus that is well known for an extreme et al., 2015). So, for our we focused on the by the from the (Fig. We the germination slightly to water from the and were to to any in the than + or – a of the was by rates and of during the of production of germination structures, formation of and of and/or of by production of and/or production of and For this a range of media were a water-activity range from to (Stevenson et al., The germination in our study indicated that be capable of germination on most of these even the three at < 0.605 water activity (Stevenson et al., Whereas there were lower water-activity of germination indicated that these would not permit germination at their respective water-activity We to work with the The who has as a to the Institute with the of strain low water-activity The to been with the of the of germination as the it that the not yet the after Andy's to Belfast. the to on media at then on some other and then by – day – there was at that is production of germination day cell at been (Fig. and well to of but the water-activity germination on a with glycerol after so of we observed and cell of a microbe at < 0.605 water in Stevenson et this finding a in the water-activity window for microbial and this is to an of the temperature window for It that the most xerophilic activity was observed on a high-glycerol although it may be that within the this In of data indicated an even lower water-activity of we up the study in which the high-glycerol system was found to be fungal germination at the water-activity limit for Stevenson et al., we focused on the biophysical role of glycerol. up the water-activity study, we the finding from an The finding has some from the and including to the COSPAR for the on the Planetary Protection for in and Planetary and to join the and and the of research The study fungi to conditions on the of the during a study et al., 2018). The has been to the research to and other microbes to conditions during a in the This is on the and thought to be the on Earth, with an temperature of and et al., et al., 2014). this may on to a study of for strain on the So, the search for an extreme xerophile was an and in to the these an extremely this fungal strain to a of and and a number of the was at values of water activity or relative humidity than or the humidity within a can often this value. However, such xerophiles can survive of and there may have been of water was once a of microbial on the of their water-activity minima that been by J. Pitt Food and which to an focused on microbes, and has been and by (e.g. a recent to I was the of a whereas the microbial was there was of based on position on the that was determined by water To me, this a living of with the microbial biosphere in – and in relation to water activity – a of environmental of water activity also occur on to such as where water is from or other including those of the The water-activity for life in of a yet one that can be and For the water of some microbial habitats that (e.g. et al., 2013; et al., can and profound in water The phenomenon occurs in on surfaces and brine systems et al., et al., 2018). microbes within may in water activity and as the the cell may be water et al., et al., This how the of a limit for life can It also that the of Earth's biosphere is as the also sampling in can microbes and The is based on work by the and Research United and of and and of and was by and Andrew Stevenson University of UK), of The University, University of Australia), of Ailsa D. Hocking Food and Nutrition, Australia), Naresh Magan (Cranfield University, England, UK), Research Research University, of N. of J. of England, UK), Kenneth N. Timmis University and Michail M. Yakimov (Institute for Coastal Marine Environment,

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