![Stress tolerance of xerophilic fungi to (A and B) single stressors and (C–I) temperature : water-activity regimes. Proportion of the 157 fungal strains tested (see Tables 1 and 2) that (A) grew optimally and (B) grew to their water-activity minimum on media containing either no added solute (control) or those supplemented with ethanol, NaCl, ethylene glycol, glycerol, MgCl2, fructose, sucrose or PEG 400. For each medium type, fungi were grown over a range of concentrations from zero (control media) to the concentration limit that prevented growth (data not shown). For three fungal strains growth-rate data obtained from single-stressor screens were plotted according to the chaotropic or kosmotropic activity of media (see later). For C–K: growth profiles for the nine most xerophilic fungi incubated at 15, 20, 25, 30 and 37°C on glycerol-supplemented media [water-activity values ranged from 0.810 to 0.653; isopleth contours indicate growth rates (mm day−1)] and were plotted using Sigmaplot, Version 8.0. The fungal strains were (C) JH06THH, (D) JH06GBM, (E) JH06GBO, (F) JH06JPD, (G) Aspergillus penicillioides FRR 2179, (H) Eurotium amstelodami FRR 2792, (I) Xeromyces bisporus FRR 0025, (J) X. bisporus FRR 3443 and (K) X. bisporus FRR 2347 (see Tables 1 and 2).](https://www.researchgate.net/profile/John-Hallsworth/publication/38021213/figure/fig2/AS:294189104418825@1447151556468/Stress-tolerance-of-xerophilic-fungi-to-A-and-B-single-stressors-and-C-I_Q320.jpg)
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Limits of life in hostile environments: no barriers to
biosphere function?emi_2079 3292..3308
Jim P. Williams and John E. Hallsworth*
School of Biological Sciences, MBC, Queen’s University
Belfast, Belfast, BT9 7BL, UK.
Summary
Environments that are hostile to life are characterized
by reduced microbial activity which results in poor
soil- and plant-health, low biomass and biodiversity,
and feeble ecosystem development. Whereas the
functional biosphere may primarily be constrained by
water activity (aw) the mechanism(s) by which this
occurs have not been fully elucidated. Remarkably we
found that, for diverse species of xerophilic fungi at
awvalues of ⱕ0.72, water activity per se did not limit
cellular function. We provide evidence that chaotro-
pic activity determined their biotic window, and
obtained mycelial growth at water activities as low as
0.647 (below that recorded for any microbial species)
by addition of compounds that reduced the net chao-
tropicity. Unexpectedly we found that some fungi
grew optimally under chaotropic conditions, provid-
ing evidence for a previously uncharacterized class
of extremophilic microbes. Further studies to eluci-
date the way in which solute activities interact to
determine the limits of life may lead to enhanced bio-
technological processes, and increased productivity
of agricultural and natural ecosystems in arid and
semiarid regions.
Introduction
Xerophilic fungi are more tolerant to water stress than any
other organism; mycelial growth of one species has been
previously recorded down to a water activity of 0.656 (Pitt
and Christian, 1968). Terrestrial fungi play key roles in the
degradation of organic matter and global nutrient cycles,
the formation and structure of soils and geological depos-
its, and via their symbiotic interactions with plants (Ruiz
and Azcon, 1995; Gunde-Cimerman et al., 2000; Jeffries
et al., 2003; Hoffland et al., 2004; van der Heijden et al.,
2008). Furthermore, the substantial fungal biomass of
soils in semiarid regions (Smith et al., 1992; Rutz and
Kieft, 2004) can act as nutrient and water reservoirs in
water-constrained ecosystems (Ruiz and Azcon, 1995;
Austin et al., 2004; Kashangura et al., 2006; Collins et al.,
2008). Xerophilic microbes have historically been isolated
and characterized in the context of food-spoilage studies
(Pitt, 1975), but they exist in nature at an indefinite
number of biosphere–environment interfaces where life is
challenged by physical and chemical barriers. Xerophilic
fungi are therefore useful model systems to investigate
the feasibility of cellular activity in arid and stressful habi-
tats (Onofri et al., 2004; Beaty and Buxbaum, 2006; Tosca
et al., 2008). Recent studies carried out on halophilic
prokaryotes and mesophilic bacterial and yeast species
from hostile environments found that chaotropicity (or
related solute activities) can limit microbial metabolism,
replication and survival (Hallsworth et al., 2003a; 2007;
Duda et al., 2004; Lo Nostro et al., 2005). For both ionic
and non-ionic solutes, neither chaotropic activities nor
Hofmeister effects is a colligative property of a solution
(see Dixit et al., 2002; Ball, 2008); furthermore the mecha-
nism of chaotropic activity for ions (see Sachs and Woolf,
2003), non-ionic solutes (see Hallsworth et al., 2003a),
and hydrophobic substances (McCammick et al., 2009; P.
Bhaganna and J.E. Hallsworth, unpublished) may differ.
Nevertheless, the Hofmeister series (for ions), chaotrop-
icity and kosmotropicity (activities of diverse chemical
species) provide frameworks that can be usefully
employed to study the affinities of substances to modify
the structural interactions of cellular macromolecules
(see Hamaguchi and Geiduschek, 1962; Collins, 1997;
Hallsworth et al., 2003a; Ball, 2008). However, such
solute activities have not been studied either at ultra-low
water activities (ⱕ0.8; see Hallsworth et al., 2007), or in
xerophilic fungi. Water activity has been highly effective in
providing a global measure of the cumulative molecular-
level, biochemical and phenotypic effects of decreased
solvent availability (see Brown, 1990; Chaplin, 2006).
Nevertheless, we recently observed that other solutes
activities, most notably chaotropicity that weakens mac-
romolecular interactions and disorders cellular structures,
can also limit biosphere function in specific localities (see
Hallsworth et al., 2007). We therefore suspected that
water activity is not a definitive parameter that dictates the
limits of microbial activity in all environmental niches. We
carried out this study of xerophiles, obtained from diverse
sources, to test the hypothesis that water activity does not
Received 8 June, 2009; accepted 27 August, 2009. *For correspon-
dence. E-mail j.hallsworth@qub.ac.uk; Tel. (+44) 28 9097 2314;
Fax (+44) 28 9097 5877. Re-use of this article is permitted in
accordance with the Terms and Conditions set out at http://
www3.interscience.wiley.com/authorresources/onlineopen.html
Environmental Microbiology (2009) 11(12), 3292–3308 doi:10.1111/j.1462-2920.2009.02079.x
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd
always act as the barrier to microbial and, by implication,
biosphere function in high-solute environments. Here we
show that in low water-activity environments that are
hostile to life (ⱕ0.72 aw), water activity per se did not limit
microbial activity; and provide evidence that cellular func-
tion was determined by the net effect of other environ-
mental parameters (including chaotropic and kosmotropic
activities) that impact on macromolecule structure-
function. We also identified a new class of extremophilic
microbe, chaophiles, that may prove to be a source of
novel enzymes for biotechnology.
Results and discussion
Xerophilic fungi from high- and low-solute substrates
We took a two-pronged approach to identifying the
ultimate, most xerophilic, microbes. First, we sampled
environments in various continents and climatic zones,
focusing our search on low-solute substrates: the sur-
faces of glass, metal, wood, leather, textiles and paper
(see Table 1; Experimental procedures). Remarkably we
found an abundance of xerophilic fungi, and isolated 107
phenotypically distinct cultures from these low-solute
environments using glycerol-supplemented (5 M glycerol;
0.845 aw) or sucrose-supplemented media (2.2 M
sucrose; 0.884 aw), predominantly on samples originating
from humid countries such as Japan, Northern Ireland
and Thailand (see Table 1). Second, we identified and
obtained cultures of the 37 most-xerophilic strains previ-
ously reported in the published literature (1900–2008); the
majority of these had been isolated from high-solute foods
(see Table 2). In addition, we contacted research groups
currently working in the field of environmental microbiol-
ogy and obtained cultures of fungi and yeasts that had
been isolated from high-salt or high-sugar environments
and/or were suspected to be highly solute-tolerant (i.e. the
14 strains from EXF and UWOPS culture collections; see
Table 2). For the purposes of the current study we used
multiple criteria to define xerophilicity: that a species must
be able to grow below 0.85 awunder at least two sets of
environmental conditions, and must also grow optimally
below 0.95 aw(see Pitt, 1975).
For all environmental isolates and the named xerophile
species (157 strains in total; see Tables 1 and 2) rates of
hyphal extension were determined on low water-activity
media containing one of a range of chemically diverse but
biologically relevant solutes (see Fig. 1A and B). Gener-
ally strains from low-solute substrates grew down to
similar water activities, and at comparable growth rates, to
those from high-solute environments (data not shown).
The solute that facilitated the optimum growth-rate varied
depending on the fungal strain, but sucrose was most
permissive for the majority of strains (Fig. 1A). By con-
trast, glycerol facilitated growth down to the lowest water-
activity for more than 75% of strains (Fig. 1B) so we used
glycerol-supplemented media to test the hypothesis that
the stress parameter water activity does not always limit
life on low water-activity substrates.
Nine out of the 157 strains grew at ⱕ0.75 aw, and these
had been isolated either from low-solute surfaces during
the current study (strains JH06THH; JH06GBM;
JH06GBO; JH06JPD from wooden surfaces, see Table 1)
or from high-solute substrates by other research groups
(strains Aspergillus penicillioides FRR 2179; Eurotium
amstelodami FRR 2792; and three strains of Xeromyces
bisporus: FRR 0025; FRR 3443; FRR 2347, see Table 2).
Mycelial growth rates of these strains were quantified on
glycerol-supplemented media over a matrix of tempera-
ture and water-activity values (Fig. 1C–K) in order to
determine the limits of their biotic windows, and to obtain
two-dimensional profiles of their growth phenotypes. We
then determined the pH required for optimum growth over
a range of water-activity values on glycerol-supplemented
media in order to avoid inadvertently causing pH limita-
tion. There were clear phenotypic differences between
strains, but growth at low water activity was generally
optimal at 30°C (see Fig. 1C–K) and pH 5.75 (data not
shown) so these conditions were used throughout the
study. Although hyphal growth has previously been
recorded at ⱕ0.710 aw(see Pitt and Christian, 1968; see
later), only two strains grew on glycerol media at water-
activity values significantly below 0.714 aw, regardless of
temperature or pH (see Fig. 1J and K). The glycerol con-
centrations used in these media (i.e. ⱕ7.16 M) are con-
sistent with the intra and/or extracellular concentrations to
which microbial cells can be exposed in nature (Brown,
1990; Hallsworth and Magan, 1994a; de Jong et al., 1997;
Hallsworth, 1998; Zhuge et al., 2001; Bardavid et al.,
2008). However, our data as well as earlier studies
suggest that glycerol has inhibitory activities at molar con-
centrations (see Fig. 1J and K; Borowitz and Brown,
1974; Hallsworth et al., 2007), and may act as a chaotro-
pic stressor due to its unusual interactions with water
and destabilizing effects on macromolecular structures
(Borowitz and Brown, 1974; Hallsworth et al., 2007; Chen
et al., 2009; F.D.L. Alves and J.E. Hallsworth, unpub-
lished). We therefore formulated the hypothesis that
solute activities other than water activity can determine
the limits of microbial-cell function.
Water activity did not limit life at low water activity
To test this hypothesis, we designed a range of 14 low
water-activity media that were supplemented with either a
chaotropic solute (fructose or glycerol) or combinations of
glycerol and a number of other solutes: fructose and/or
the kosmotropes sucrose, glucose, NaCl and KCl (Collins,
Limits of life in hostile environments 3293
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology,11, 3292–3308
Table 1. Fungal strains isolated from diverse substrates during the current study.a
Strain
designationbEnvironmental source (country)
Strain
designationbEnvironmental source (country)
JH05GB42 Copper pipe in 12°C constant-temperature room (UK) JW07JP14 Dead bamboo (Japan)
JH05GB43 Copper pipe in 12°C constant-temperature room (UK) JW07JP18 Surface of firewood in outdoor woodpile (Japan)
JH06GBa Underside of an antique earthenware-bowl (UK) JW07JP20 External wall of a wooden hut (Japan)
JH06GBb Dust on the floor of a living room (UK) JW07JP21 Insect pupa (Japan)
JH06GBc Blue (Stilton) cheese (UK) JW07JP25 Surface of firewood in outdoor woodpile (Japan)
JH06GBB Stem of dried protea flower (South Africa) JW07JP29 Aluminium windowsill on the outside of a building
(Japan)
JH06GBl Paint work of a 1922 wooden window-frame (UK) JW07JP30a Aluminium windowsill inside a building (Japan)
JH06GBM Underside of an antique sycamore chopping-block
(UK)
JW07JP30b Aluminium windowsill inside a building (Japan)
JH06GBN Underside of an antique sycamore chopping-block
(UK)
JW07JP30c Aluminium windowsill inside a building (Japan)
JH06GBO Underside of an antique sycamore chopping-block
(UK)
JW07JP36 Glass surface of a window inside a building (Japan)
JH06GBW Antique felt (UK) JW07JP41a Wooden floor (Japan)
JH06IL49 Semi-dried date (Israel) JW07JP41b Wooden floor (Japan)
JH06IL50 Semi-dried date (Israel) JW07JP43 Old glass light-bulb (Japan)
JH06IN45 Semi-dried tamarind pods (India) JW07JP49 Underside of a stone table – outdoors (Japan)
JH06IN46 Semi-dried tamarind pods (India) JW07JP51 Surface of wooden bench – outdoors (Japan)
JH06IN47 Antique wooden artefact (India) JW07JP61 Rotting wood (Japan)
JH06IN48 Antique wooden artefact (India) JW07JP64 Dead tree-trunk (Japan)
JH06JPj Antique wooden artefact (Japan) JW07JP74 Aluminium windowsill inside a building (Japan)
JH06JPD Antique wooden rice-scoop (Japan) JW07JP75 Old cotton cushion-cover (Japan)
JH06JPE Inner surface of an antique bronze bell (Japan) JW07JP83 Tree trunk (Japan)
JH06JPF Inner surface of an antique bronze bell (Japan) JW07JP95 Surface of wooden bench – outdoors (Japan)
JH06JPQ Antique wooden rice-pot lid (Japan) JW07JP96 Stone table – outdoors (Japan)
JH06JPS Antique wooden rice-pot lid (Japan) JW07JP99 Underside of a wooden bench – outdoors (Japan)
JH06JPT Antique wooden rice-pot lid (Japan) JW07JP117a Internal surface of dried bamboo (Japan)
JH06NAV Stem of a wild grape (Namibia) JW07JP117b Internal surface of dried bamboo (Japan)
JH06THH Antique wooden artefact (Thailand) JW07JP120a Antique wooden artefact (Japan)
JH06THI Antique wooden artefact (Thailand) JW07JP120b Antique wooden artefact (Japan)
JH06THJ Antique wooden artefact (Thailand) JW07JP160 Antique wooden artefact (Japan)
JH06THK Antique wooden artefact (Thailand) JW07JP166 Rotting bamboo (Japan)
JH06ZA44 Grass basket (South Africa) JW07JP167 Rotting bamboo (Japan)
JH06ZA51 Tin surface of a food can (South Africa) JW07JP168a Rotting bamboo (Japan)
JH06ZA52 Tin surface of a food can (South Africa) JW07JP168b Rotting bamboo (Japan)
JH06ZAU Glass of a 1940’s picture frame (South Africa) JW07JP169 Rotting bamboo (Japan)
JH07JP126 Antique bronze vase (Japan) JW07JP170a Rotting bamboo (Japan)
JH07JP127 Green leaf (Japan) JW07JP170b Rotting bamboo (Japan)
JH07JP128 Old earthenware bonsai-container (Japan) JW07JP171a Rotting bamboo (Japan)
JH07JP130 Green bamboo (Japan) JW07JP171b Rotting bamboo (Japan)
JH07JP133 Rotting wood (Japan) JW07JP172 Rotting bamboo (Japan)
JH07JP138 Old cedarwood-container (Japan) JW07JP173 Old, dried Reiki mushroom (Japan)
JH07JP141 Bamboo leaf (Japan) JW07JP174 Old, dried Reiki mushroom (Japan)
JH07JP143 Green bamboo (Japan) JW07JP175a Old, dried Reiki mushroom (Japan)
JH07JP144 Leaf surface (Japan) JW07JP175b Old, dried Reiki mushroom (Japan)
JH07JP146 Dead bamboo (Japan) JW07JP176 Old, dried Reiki mushroom (Japan)
JH07JP148 Rotting bamboo (Japan) JW07JP177 Old, dried Reiki mushroom (Japan)
JH07JP149 Rotting bamboo (Japan) JW07JP179 Old, dried Reiki mushroom (Japan)
JH07JP151 Rotting leaf (Japan) JW07JP180 Moulding surface of tree branch (Japan)
JH07JP154 Wooden bathroom wall (Japan) JW07JP181 Moulding surface of bamboo (Japan)
JH07JP156 Wooden bathroom wall (Japan) JW07JPc118 Airborne spores (Japan)
JH07ZA147 Wooden artefact (South Africa) RS07PT1 Laboratory contaminant (Portugal)
JW07GB158 Antique mahogany table-top (UK) RS07PT2 Laboratory contaminant (Portugal)
JW07JP2 Metal surface of an armrest on a 1970’s train (Japan) RS07PT3 Laboratory contaminant (Portugal)
JW07JP4 Silicon floor-seal on a 1970’s train (Japan) RS07US5 Soil (North America)
JW07JP8 Silk toy hung on exterior of a building (Japan) RS07US10 Soil (North America)
JW07JP13 Insect faeces on dead bamboo (Japan)
a. Strains were isolated on glycerol-supplemented and sucrose-supplemented MYPiA medium; see Experimental procedures. Strains RS07PT1,
RS07PT2, RS07PT3, RS07US5 and RS07US10 were isolated by Ricardo dos Santos, Laboratório de Análises of the Instituto Superior Técnico,
Portugal. Entries in bold correspond to strains selected for more detailed study (see Figs 1C–K, 2 and 3A).
b. The third and fourth characters of strain designations indicate the year that sampling and isolation were carried out (i.e. 2005, 2006 or 2007).
3294 J. P. Williams and J. E. Hallsworth
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology,11, 3292–3308
Table 2. Named xerophilic and solute-tolerant species that were used in the current study.a
Species Strain designation Environmental source (country) Relevant reference(s)
Aspergilus glaucus IMI 053242 Microscope objective (Sri Lanka)
Aspergilus nidulans var. echinulatus CBS 120.55; IMI 061454ii Not stated (Argentina) Fennell and Raper (1955)
Aspergilus penicillioides ATTC 14567; FRR 3735 Binocular lens (Australia)
A. penicillioides ATTC 16910; FRR 3722 Human lobomycosis (Australia) Gock et al. (2003)
A. penicillioides FRR 2179 Dried chillies (Australia)
A. penicillioides FRR 3795 Audio tape (Australia)
Aspergillus wentii CBS 104.07; IMI 017295ii Soybeans (Indonesia)
Basipetospora chlamdospora IMI 332258 Soil (Chile)
Brettanomyces bruxellensis UWOPS 94-239.3 Tequila fermentation (Mexico)
Candida apicola UWOPS 01-663b2 Merremia tuberosa flower (Costa Rica)
Candida berthetii ATCC 18808; CBS 5452 Arabic gum (Cameroon) Boidin et al. (1963)
Candida etchellsii UWOPS 01-168.3 Bee hive (Costa Rica)
Candida hawaiiana UWOPS 04-206.8 Prosopeus cf. bidens (nitidulid beetle) from llex anomala flower (Hawaii)
Chrysosporium fastidium ATTC 18053; FRR 0077 Improperly sundried prunes (Australia) Hocking and Pitt (1980); Pitt and Hocking (1977)
C. fastidium FRR 0081 Dried prunes (Australia)
Chrysosporium xerophilium ATTC 18052; FRR 0530 High-moisture prunes (Australia) Kinderlerer (1995)
Cladosporium sphaerospermum EXF 738 Bathroom (Slovenia) Zalar et al. (2007)
Debaryomyces hansenii DSMZ 3428 Spoilt sake (not stated)
D. hansenii DSMZ 70590 Harzer cheese
D. hansenii UWOPS 05-230.3 Beetle, Bertam Palm (Malaysia)
Debaryomyces melissophilus UWOPS 01-677c6 Conotelus (nitidulid beetle) from M. tuberosa flower (Costa Rica)
Eurotium amstelodami ATTC 16464; FRR 2792 Dates (Australia) Tamura et al. (1999)
E. amstelodami ATTC 42685; FRR 0475 Dried prunes (Australia) Hocking and Pitt (1980)
Eurotium chevalieri ATTC 28248; FRR 1311 Spoiled prunes (Australia) Pitt and Hocking (1977)
Eurotium echinulatum FRR 2419 Hazelnut kernels (Australia)
E. echinulatum FRR 5040 Sultanas (Australia)
Eurotium halophilicum ATTC 62923; FRR 2471 Cardamom seeds (Australia) Hocking and Pitt (1988)
Eurotium herbariorum FRR 2418 Hazelnut kernels (Australia)
E. herbariorum FRR 5004 Sultanas (Australia)
E. herbariorum FRR 5354 Liquorice (Australia)
Hortaea werneckii EXF 225 Hypersaline saltern (Slovenia)
Kodamaea ohmeri UWOPS 05-228.2 Beetle, Bertam Palm (Malaysia)
Pichia sydowiorum UWOPS 03-414.2 Nectar, Bertam Palm (Malaysia)
Polypaecilum pisce FRR 2732; IMI 288726ii Dried fish (Indonesia)
Saccharomyces cerevisae CCY 21-4-13 Not stated (not stated)
Saccharomyces ludwigii UWOPS 92-218.4 Tequila fermentation (Mexico)
Starmerella bombicola UWOPS 01-123.1 Bee from Ipomoea trifida (Costa Rica)
Wallemia ichthyophaga CBS 818.96 Sunflower seed (Sweden) Vaupotic and Plemenitas (2007); Zalar et al. (2005)
Wallemia muriae MZKI B-952 Hypersaline saltern (Slovenia) Vaupotic and Plemenitas (2007); Zalar et al. (2005)
Wallemia sebi EXF 994 Hypersaline saltern (Slovenia) Zalar et al. (2005)
W. sebi EXF 1053 Dead Sea (Israel) Zalar et al. (2005)
W. sebi FRR 4623 Maple syrup (Australia)
Xeromyces bisporus ATTC 28298; FRR 0025 High-moisture prunes (Australia)
X. bisporus ATTC 36964; FRR 1522 Spoiled liquorice (Australia) Hocking and Pitt (1980); Pitt and Hocking (1977)
X. bisporus FRR 2347 Fruit cake (Australia) Gock et al. (2003)
X. bisporus FRR 3443 Raisins (Australia)
X. bisporus IMI 317902 Chinese dates (Australia)
Zygosaccharomyces rouxii ATTC 28166; FRR 3669 Table wine (Australia) Andrews and Pitt (1987)
Z. rouxii FRR 3681 Fructose corn-syrup (Australia) Andrews and Pitt (1987)
Z. rouxii FRR 5304; NCYC 381 Sugarcane (Australia) Corry (1976)
a. Cultures were obtained from the American Type Culture Collection (ATTC, USA), the Centraalbureau voor Schimmelcultures (CBS, Netherlands), the Culture Collection of Yeasts (CCY,
Slovakia), the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany), the Extremophilic Fungi Culture Collection (EXF, Slovenia), the Food Research Ryde (FRR, Australia),
the International Mycological Institute (IMI, UK), the Microbial Culture Collection of National Institute of Chemistry (MZKI, Slovenia), the National Collection of Yeast Cultures (NCYC, UK), and the
University of Western Ontario Plant Sciences Culture Collection (UWOPS, Canada).
Limits of life in hostile environments 3295
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology,11, 3292–3308
AB
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Water activity
Temperature (˚C)
3296 J. P. Williams and J. E. Hallsworth
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology,11, 3292–3308
1997; Galinski et al., 1997; see Table 3 and Fig. 2A–I). A
number of recent studies provide evidence that some ions
can penetrate the hydrophobic domains of macromolecu-
lar systems by shedding their hydration water and that, via
their physical bulk, they disorder the tertiary/quaternary
structure (see Sachs and Woolf, 2003), i.e. that – by our
earlier definition – they act chaotropically. However, NaCl
and KCl that were used to depress water activity in Media
7, 9 and 12 are kosmotropic (i.e. solutions of their ions
have a net kosmotropic activity; see Table 3), and this is
consistent with their stabilizing effects on membranes,
proteins and other cellular structures (see Brown, 1990).
The range of water-activity values tested (0.760–0.644 aw)
lies at the extreme edge of the water-activity window for
all nine xerophile strains under study, and growth optima
at 30°C lay between 0.95 and 0.85, as shown for one
X. bisporus strain in Fig. 2J. Remarkably there was no
correlation between rates of radial extension for these
nine fungi (the most xerophilic microbes thus far identi-
fied) on glycerol-containing media at ⱕ0.72 aw, and the
water activity of their culture media (Fig. 2A–I). Generally,
on glycerol-supplemented media at ⱕ0.85 aw, the growth
rates of all strains decreased in proportion to medium
water activity (e.g. see Fig. 2J). On the glycerol-
supplemented medium at 0.644 awand the fructose-
supplemented medium at 0.760 aw(despite the relatively
high water activity of the latter; see Table 3) there was no
hyphal growth of any xerophile strain (therefore data are
not shown for Media 13 or 14 in Fig. 2). Paradoxically, for
Medium 1 (at 0.714 aw) eight out of the nine fungal strains
either failed to grow (Fig. 2A–D) or grew 65–90% more
slowly than predicted (see Fig. 2E–G and I), whereas at
lower water-activities (0.670–0.647) growth rates were up
to 580% greater than predicted (i.e. the mixed-solute
Media 6–9 and 12; see Fig. 2A–I).
The lowest water activity previously reported for sus-
tained growth of fungi was 0.656: for X. bisporus after a
90 day incubation period (Pitt and Christian, 1968). By
comparison several fungal strains grew in the current
study at 0.656 aw, and hyphal growth was observed at this
water activity for one strain after only 11 days (Fig. 2F;
Table 4). Remarkably we observed growth at water-
activity values as low as 0.647, and did so in as little as
5–8 weeks, on mixed-solute media (see Fig. 2A–I;
Table 4). Furthermore, four out of the five strains that were
able to grow at 0.647 awhad been isolated from low-solute
surfaces (in the current study; see Table 1) and were
therefore more xerophilic than all but one of the strains
Fig. 1. Stress tolerance of xerophilic fungi to (A and B) single stressors and (C–I) temperature : water-activity regimes. Proportion of the 157
fungal strains tested (see Tables 1 and 2) that (A) grew optimally and (B) grew to their water-activity minimum on media containing either no
added solute (control) or those supplemented with ethanol, NaCl, ethylene glycol, glycerol, MgCl2, fructose, sucrose or PEG 400. For each
medium type, fungi were grown over a range of concentrations from zero (control media) to the concentration limit that prevented growth (data
not shown). For three fungal strains growth-rate data obtained from single-stressor screens were plotted according to the chaotropic or
kosmotropic activity of media (see later). For C–K: growth profiles for the nine most xerophilic fungi incubated at 15, 20, 25, 30 and 37°C on
glycerol-supplemented media [water-activity values ranged from 0.810 to 0.653; isopleth contours indicate growth rates (mm day-1)] and were
plotted using Sigmaplot, Version 8.0. The fungal strains were (C) JH06THH, (D) JH06GBM, (E) JH06GBO, (F) JH06JPD, (G) Aspergillus
penicillioides FRR 2179, (H) Eurotium amstelodami FRR 2792, (I) Xeromyces bisporus FRR 0025, (J) X. bisporus FRR 3443 and (K)
X. bisporus FRR 2347 (see Tables 1 and 2).
Table 3. Chaotropic-activity and water-activity values for solutes and solute combinations used to supplement growth media.a
Medium
designationa
Added solute(s); concentration [M]
Chaotropic activity (kJ kg–1)b
Water
activityc
Glycerol NaCl KCl Fructose Glucose Sucrose
1 6.84 0 0 0 0 0 Highly chaotropic (15.27) 0.714
2 7.06 0 0 0 0 0 Highly chaotropic (16.64) 0.702
3 5.34 0 0 0 0 0.73 Relatively neutral (12.48d) 0.699
4 7.48 0 0 0 0 0 Highly chaotropic (18.05) 0.686
5 7.48 0 0 0 0 0 Highly chaotropic (18.05) 0.685
6 5.97 0 0 0 0 0.73 Relatively neutral (11.11d) 0.670
7 3.91 1.20 0.13 0 0 0.73 Relatively neutral (-2.75d,e) 0.667
8 4.34 0 0 1.11 1.11 0 Relatively neutral (9.73d) 0.665
9 4.67 1.20 0.13 0 0 0.73 Relatively neutral (-2.75d,e) 0.656
10 7.60 0 0 0 0 0 Highly chaotropic (28.80) 0.655
11 7.60 0 0 0 0 0 Highly chaotropic (20.80) 0.653
12 6.19 1.20 0.13 0 0 0 Relatively neutral (2.79d) 0.647
13 7.65 0 0 0 0 0 Highly chaotropic (20.88) 0.644
14 0 0 0 4.80 0 0 Highly chaotropic (20.80) 0.760
a. See Fig. 2A–I. The pH of all media was 5.75, except for Medium 4 (pH 4).
b. See Hallsworth and colleagues (2003a).
c. Measured at 30°C.
d. Extrapolated from agar gel-point curve.
e. Media were slightly kosmotropic so the activity value is negative.
Limits of life in hostile environments 3297
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology,11, 3292–3308
0.06
0.09
0.12
0.15
0.18
0.21
0.24
0.27
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.27
0.36
0.45
BA C
0.36
0.48
0.60
0.27
0.36
0.45
0.06
0.08
0.10
0.12
0.00
0.03
0.06
0.00
0.02
0.00
0.09
h rate (mm d-1)
DEF
0.16
0.20
0.36
0.42
0.20
0.24
0.00
0.12
0.24
0.00
0.09
0.18
0.00
0.02
0.04
Radial growth
IHG
0.00
0.04
0.08
0.12
7)
3)
5)
6)
5)
7)
0)
5)
6)
9)
2)
4)
0.00
0.06
0.12
0.18
0.24
0.30
7)
3)
5)
6)
5)
7)
0)
5)
6)
9)
2)
4)
0.00
0.04
0.08
0.12
0.16
7)
3)
5)
6)
5)
7)
0)
5)
6)
9)
2)
4)
12 (0.64
11 (0.65
10 (0.65
9 (0.65
8 (0.66
7 (0.66
6 (0.67
5 (0.68
4 (0.68
3 (0.69
2 (0.70
1 (0.714
12 (0.647
11 (0.653
10 (0.655
9 (0.656
8 (0.665
7 (0.667
6 (0.670
5 (0.685
4 (0.686
3 (0.699
2 (0.702
1 (0.714
12 (0.64
11 (0.65
10 (0.65
9 (0.65
8 (0.66
7 (0.66
6 (0.67
5 (0.68
4 (0.68
3 (0.69
2 (0.70
1 (0.71
Media type (water activity)
1)growth rate (mm d-1
Water activity
Radial g
3298 J. P. Williams and J. E. Hallsworth
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology,11, 3292–3308
isolated from high-solute environments during the past
100 years (see Fig. 2A–I; Table 4). One fungal strain, iso-
lated in 2006 from the wooden (sycamore) surface of a
19th Century, kitchen chopping-block (JH06NIM; see
Table 1), was observed to be growing at 0.647 awafter
only 34 days incubation at 30°C (Fig. 2B; Table 4).
For a given fungal species, the lower water-activity limit
for the germination of propagules is typically lower than
that for hyphal growth (Pitt, 1975). However, numerous
studies of spore germination of xerophiles at low water-
activities (see Table S1) have found that growth ceases
upon germ-tube production (Pitt and Christian, 1968).
Whereas a number reviews cite germ-tube formation by
X. bisporus spores at 0.605 awas evidence of cellular
function at ultra-low water activity (Pitt, 1975; Grant, 2004;
see also Table S1), further hyphal growth and mycelium
development were not recorded (Pitt and Christian, 1968).
In the current study hyphal growth of A. penicillioides and
E. amstelodami occurred at considerably lower water-
activity values (0.647 and 0.656 awon mixed-solute
media; see Fig. 2E and F; Table 4) than those previously
reported for germination (i.e. 0.680 and 0.703 respec-
tively; see Table S1). For each xerophile strain at water-
activity values below their growth optimum, growth rates
were proportionally reduced (for an example, see Fig. 2J).
However, at extremely low water activity (ⱕ0.72 aw),
growth rates were no longer proportional to water activity
so we concluded that other stress parameters limited
cellular activity. Furthermore, we asked the scientific
questions whether the chaotropicity of glycerol-
supplemented media (6.84–7.65 M glycerol) limited
hyphal growth at low water activity, and whether this
inhibition was reversed by the kosmotropic activity of
other substances present in the mixed-solute media
(3.91–6.19 M glycerol).
Chaotropic compounds limited cell function, but their
effects were reversible
The fructose-supplemented medium and the seven
glycerol-supplemented media were found to have
chaotropic-activity values of 15–21 kJ kg solution-1
(Table 3); values that are consistent with the chaotropicity
limits for other microbial species (see Hallsworth et al.,
2007). However, the activity values of the six mixed-solute
media that contained kosmotropes were relatively neutral
(12.48 to -2.75 kJ kg solution-1; see Table 3). Generally
there were either low rates of radial extension on chao-
tropic media, or no growth at all (for glycerol-only media
see Fig. 2A–I, orange columns; for fructose-only media
data not shown). By contrast, remarkably high growth-rate
values were obtained on the other media that were neutral
or mildly chaotropic, and these were several hundred per
cent higher than those predicted from water-activity
values (see Fig. 2A–I, black columns). There was a strong
inverse correlation between chaotropic activity and fungal
growth (see Figs 2A–I and 3A): three strains that were
able to grow down to 0.647 awdid not grow on any
glycerol-supplemented media even at the relatively less-
stressful water activity of 0.714 (Fig. 2A–C).
Glycerol, which is neutral or only weakly chaotropic
below concentrations of 3–4 M (F.D.L. Alves and J.E.
Hallsworth, unpublished), is widely known for its activities
as a stress protectant that can both protect the structure
and function of cellular macromolecules, and act as an
intracellular osmolyte to control cell turgor (Brown, 1990;
Dashnau et al., 2006). At higher concentrations (ⱖ6M),
however, we have demonstrated the extreme chaotro-
picity of glycerol (Table 3). On high-solute substrates
xerophile cells can fail due to the prohibitive energy
expenditure required to retain the intracellular glycerol
that is needed as an osmolyte (Hocking, 1993). We
propose that glycerol itself can disorder and permeabilize
the plasma membrane, via its chaotropic activity, thereby
resulting in the leakage of this protectant from the cell.
Whereas the biochemical mechanisms by which chao-
tropic solutes disorder cellular macromolecules are not
yet fully understood (see above), we have illustrated the
structural consequences for macromolecular systems in a
cell stressed by a chaotropic solute, and the way in which
kosmotropic solutes counter chaotrope-induced stress
(Fig. 3B–E). For a cell growing under optimal conditions
Fig. 2. Growth rates at 30°C (A–I) for the nine selected xerophiles (see also Fig. 1C– K) on highly chaotropic (solid-orange columns) or
neutral media (black columns) over a range of water-activity values (0.714–0.647; see also Table 3): Medium (1) glycerol (6.84 M), Medium (2)
glycerol (7.06 M), Medium (3) glycerol (5.43 M), sucrose (0.73 M) plus NaNO3(0.24 M), Medium (4) glycerol (7.48 M, and concentrations of
malt extract, yeast extract and K2HPO4that were 10-fold more dilute than those of the control medium), Medium (5) glycerol (7.48 M), Medium
(6) glycerol (5.97 M), sucrose (0.73 M), Medium (7) glycerol (3.91 M), sucrose (0.73 M), NaCl (1.20 M) plus KCl (0.13 M), Medium (8) glucose
(1.11 M), glycerol (4.34 M) plus fructose (1.11 M), Medium (9) glycerol (4.67 M), sucrose (0.73 M), NaCl (1.20 M) plus KCl (0.13 M), Medium
(10) glycerol (7.60 M, pH 4), Medium (11) glycerol (7.60 M), and Medium (12) glycerol (6.19 M), NaCl (1.20 M) plus KCl (0.13 M). The pH of all
media was 5.75 unless otherwise stated; chaotropic-activity values are shown in Table 3; the experiment was conducted on three independent
occasions and variation of growth-rate values was within ⫾0.2 mm day-1. Growth rates are shown for the following fungal strains: (A)
JH06THH, (B) JH06GBM, (C) JH06GBO, (D) JH06JPD, (E) Aspergillus penicillioides FRR 2179, (F) Eurotium amstelodami FRR 2792, (G)
Xeromyces bisporus FRR 0025, (H) X. bisporus FRR 3443 and (I) X. bisporus FRR 2347 (see also Fig. 1C– K; Tables 1 and 2). Theoretical
growth-rate values that were predicted based on the assumption that growth rates are proportional to medium water activity are shown for
highly chaotropic media as shaded yellow columns and for neutral media as shaded grey columns.
J. Growth curve of X. bisporus FRR 0025 over a full range of water activity values, showing the position of G (inset, lower right) in the context
of the entire biotic window of this strain on glycerol-supplemented media at 30°C (see also Fig. 1I).
Limits of life in hostile environments 3299
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology,11, 3292–3308
3300 J. P. Williams and J. E. Hallsworth
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology,11, 3292–3308
macromolecular structures and activities will presumably
be optimal (see Fig. 3B). We propose that a glycerol-
stressed cell, like those on Media 1, 2, 4, 5, 10, 11 in
Fig. 2A–I, has disordered macromolecular and membrane
structures, and increased membrane permeability (see
Fig. 3C). Conversely, a kosmotrope-stressed cell, such as
those on high-sucrose media, is likely to have highly
ordered macromolecular structures (e.g. a rigidified
plasma membrane), reduced membrane permeability,
and may also be osmotically stressed (see Fig. 3D). We
propose that a cell simultaneously exposed to opposing
chaotropic and kosmotropic activities (such that the net
effect is close to neutral; Media 3, 6, 7, 8, 9, 12 in
Fig. 2A–I; Table 3), has macromolecular structures that
are neither highly disordered nor highly ordered (Fig. 3E).
Superficially, the appearance of macromolecular struc-
tures in an optimally growing cell and a stressed cell
exposed to a chaotrope-kosmotrope mixture (Media 3, 6,
7, 8, 9, 12 in Fig. 2A–I) are qualitatively similar (Fig. 3B
and D). However, the metabolic activity and growth rate of
the latter can be orders of magnitude lower (see Fig. 2J)
than that of the optimally growing cell illustrated in Fig. 3B,
because it is subjected to the inhibitory effects of solutes
such as lowered intra/extracellular water activity and/or
solute-crowding effects, and furthermore the opposing
chaotropic/kosmotropic solute activities may not be
evenly balanced.
In summary, the data presented here (Figs 1, 2 and 3A;
Table 3) support the hypothesis that the chaotropic activ-
ity of glycerol, not the stress parameter water activity,
limits cell metabolism for these xerophilic fungi at ⱕ0.72
aw. This is consistent with evidence from other reports that
chaotropicity limits microbial function (Hallsworth, 1998;
2003a; Duda et al., 2004; Lo Nostro et al., 2005), with
a study showing that compatible solutes can reduce
ethanol stress in conidia of Aspergillus nidulans
(Hallsworth et al., 2003b), and with our recent studies of
halophilic prokaryotes which demonstrated that the
macromolecule-structuring activities of kosmotropic salts
can reduce or reverse the inhibitory effects of chaotropic
salts (Hallsworth et al., 2007).
Chaophiles: a new class of stress-tolerant organism
There are several classes of solute-tolerant microbe:
salt-tolerant halophiles, sugar-tolerant osmophiles, and
xerophiles that tolerate low water activity (whereas con-
ceptually distinct these ecophysiological groupings may
not be mutually exclusive; see Brown, 1990). Although we
recently proposed a new ecophysiological grouping,
chaophilic microbes, a search for chaotrope-tolerant
strains in samples taken from the hypersaline deep-sea
Discovery Basin proved fruitless (Hallsworth et al., 2007).
The current study provides the first evidence, to our
knowledge, for an apparent chaotropicity preference of
physiologically active cells (see Fig. 2G–I; Table 4).
Four strains failed to grow on any chaotropic media
(Fig. 2A–D), however, strains of X. bisporus were able to
tolerate all highly chaotropic media (up to 7.60 M glycerol;
Fig. 2G–I). For example, X. bisporus strain FRR 3443
grew fastest under chaotropic conditions (on Media 1 and
2), even grew at 0.653 awon a chaotropic glycerol-
supplemented medium (Medium 11), and failed to grow on
the three mixed-solute media that were either very weakly
chaotropic or slightly kosmotropic (Media 3, 7 and 9, see
Fig. 2H; Table 3). Whereas this strain also grew on Media
6 and 8 (Fig. 2H) that were relatively neutral, it is note-
worthy that these two media were more chaotropic (11.11
and 9.73 kJ kg-1respectively) than the majority of the
other media allocated to the neutral or slightly kosmotro-
pic categories (Table 3). High temperatures, like chaotro-
pic substances, disorder cellular membranes and other
macromolecular structures (Hallsworth et al., 2003a), and
growth of X. bisporus strains on high-glycerol media was
optimal at 30°C (Fig. 1I–K), but at 22°C for the other
xerophile strains (Fig. 1C-H). Although X. bisporus strains
were apparently more xerophilic at higher temperature,
this phenotype may actually indicate a preference for
conditions that disorder macromolecular and membrane
structures. Collectively, these data provide evidence for a
new class of extremophilic microbes that are chaotolerant
or chaophilic.
Implications and conclusions
We already have an understanding of environmentally
relevant solute stresses [osmotic stress (Dutrochet,
1826), matric stress (Griffin, 1977) and chaotrope-
induced water stress (Hallsworth et al., 2003a)]; how
chaotropic agents determine the limits of macromolecule
function (see Hallsworth et al., 2003a; 2007; Duda et al.,
2004); indications of the cellular components that fail
Fig. 3. Growth rates of three representative xerophilic fungi (A); Xeromyces bisporus FRR 3443, Eurotium amstelodami FRR 2792, and
isolate JH06THAJ (see Tables 1 and 2) in relation to chaotropic and kosmotropic activities of culture media: (I) NaCl (4.28 M, 0.775 aw), (II)
NaCl (3.59 M, 0.812 aw), (III) sucrose (2.34 M, 0.831 aw), (IV) PEG 400 (1.25 M, 0.855 aw), (V) glycerol (4.90 M, 0.828 aw), (VI) fructose
(3.51 M, 0.829 aw), (VII) fructose (3.94 M, 0.804 aw), (VIII) fructose (4.36 M, 0.791 aw), (IX) glycerol (6.66 M, 0.747 aw), (X) ammonium nitrate
(4.30 M, 0.855 aw) and (XI) ammonium nitrate (5.15 M, 0.817 aw). Values are means of three replicates and bars represent standard errors.
The data approximate to a Normal distribution (see dotted line), although it may be that the osmotic stress or other stress parameters
associated with kosmotropic stressors ultimately limit hyphal growth. Diagrammatic illustrations (B–E) of the way in which chaotropic and
kosmotropic activities impact on macromolecule and membrane structure in relation to an unstressed cell (B); in a chaotrope (e.g.
urea)-stressed cell (C), a kosmotrope (e.g. sucrose)-stressed cell (D), and a cell exposed to both chaotropes and kosmotropes (E).
Limits of life in hostile environments 3301
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology,11, 3292–3308
Table 4. Fungal strains capable of hyphal growth ⱕ0.71 water activity.a
Species and/or strain designation
Nature of
substrate
of originb
Lowest recorded
water activity for
hyphal growthc
Earliest
observation
of hyphal
growth (day)
Rate of
hyphal
extension
(mm day-1) Method used to reduce water activity (referenced)
Chaotropic or kosmotropic
activity of culture
medium (kJ kg-1)
JH06GBM L-S 0.647 34 0.05 Glycerol (6.19 M), NaCl (1.20 M), KCl (0.13 M)eRelatively neutral (2.79)
Aspergillus penicillioides FRR 2179 H-S 0.647 46 0.13 Glycerol (6.19 M), NaCl (1.20 M), KCl (0.13 M)eRelatively neutral (2.79)
JH06THH L-S 0.647 46 0.06 Glycerol (6.19 M), NaCl (1.20 M), KCl (0.13 M)eRelatively neutral (2.79)
JH06GBO L-S 0.647 60 0.06 Glycerol (6.19 M), NaCl (1.20 M), KCl (0.13 M)eRelatively neutral (2.79)
JH06THJ L-S 0.647 60 0.03 Glycerol (6.19 M), NaCl (1.20 M), KCl (0.13 M)eRelatively neutral (2.79)
Xeromyces bisporus FRR 3443 H-S 0.653 41 0.03 Glycerol (7.60 M)eHighly chaotropic (20.80)
X. bisporus FRR 2347 H-S 0.653 41 0.01 Glycerol (7.60 M)eHighly chaotropic (20.80)
X. bisporus FRR 1522 H-S 0.653 41 0.01 Glycerol (7.60 M)eHighly chaotropic (20.80)
Eurotium amstelodami FRR 2792 H-S 0.656 11 0.12 Glycerol (4.67 M), sucrose (0.73 M), NaCl (1.20 M),
KCl (0.13 M)e
Relatively neutral (-2.75)f
X. bisporus FRR 0025 H-S 0.656 22 0.39 Glycerol (4.67 M), sucrose (0.73 M), NaCl (1.20 M),
KCl (0.13 M)e
Relatively neutral (-2.75)f
A. penicillioides FRR 3722 H-S 0.656 29 0.13 Glycerol (4.67 M), sucrose (0.73 M), NaCl (1.20 M),
KCl (0.13 M)e
Relatively neutral (-2.75)f
E. amstelodami FRR 0475 H-S 0.656 29 0.12 Glycerol (4.67 M), sucrose (0.73 M), NaCl (1.20 M),
KCl (0.13 M)e
Relatively neutral (-2.75)f
JH06THI L-S 0.656 60 0.03 Glycerol (4.67 M), sucrose (0.73 M), NaCl (1.20 M),
KCl (0.13 M)e
Relatively neutral (-2.75)f
X. bisporus H-S 0.656 90 Not quantified Thin layer of medium on a glass surface in a
humidity-controlled chamber (Pitt and Christian, 1968)g
Not quantified
X. bisporus H-S 0.663 120 Not quantified Thin layer of medium on a glass surface in a
humidity-controlled chamber (Pitt and Christian, 1968)g
Not quantified
JH06JPD L-S 0.667 11 0.10 Glycerol (3.91 M), sucrose (0.73 M), NaCl (1.20 M),
KCl (0.13 M)e
Relatively neutral (-2.75)f
JH07JP128 L-S 0.667 94 0.02 Glycerol (3.91 M), sucrose (0.73 M), NaCl (1.20 M),
KCl (0.13 M)e
Relatively neutral (-2.75)f
Eurotium halophilicum FRR 2471 H-S 0.675 38 Not quantified Equal weights of glucose and fructose added to growth
media (Andrews and Pitt, 1987)h
Not quantified
Chrysosporium xerophilium FRR 0530 H-S 0.686 118 0.01 Glycerol (7.48 M)eHighly chaotropic (18.05)
Chrysosporium fastidium H-S 0.697 64 Not quantified Thin layer of medium on a glass surface in a
humidity-controlled chamber (Pitt and Christian, 1968)g
Not quantified
C. xerophilium H-S 0.708 80 Not quantified Thin layer of medium on a glass surface in a
humidity-controlled chamber (Pitt and Christian, 1968)g
Not quantified
Eurotium chevalieri PIL 119 H-S 0.710 16 0.1 A thin layer of medium enclosed in a humidity-controlled,
bung-sealed glass test tube (Ayerst, 1969)i
Not quantified
E. amstelodami PIL 120 H-S 0.710 32 0.1 A thin layer of medium enclosed in a humidity-controlled,
bung-sealed glass test tube (Ayerst, 1969)j
Not quantified
a. Data for the yellow-shaded entries were obtained from the current study.
b. H-S =isolated from a high-solute substrate; L-S =isolated from a low-solute surface.
c. Compiled using data from the current study and from published xerophile studies; refs. Pitt and Christian (1968); Ayerst (1969); Andrews and Pitt (1987).
d. Data were obtained from the current study unless otherwise stated.
e. The culture medium was MYPiA (pH 5.75, 30°C); see Experimental procedures.
f. N.B. Media were slightly kosmotropic so the activity value is negative.
g. The culture medium was Czapek Invert Malic Agar (pH 3.8, 25°C).
h. The culture medium was Yeast Nitrogen Base +2% glucose w/v +2% agar w/v.
i. The culture medium was Malt Extract Agar; MEA (30–40°C).
j. The culture medium was MEA (24–30°C).
3302 J. P. Williams and J. E. Hallsworth
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology,11, 3292–3308
under extreme forms of stress (see current study;
Hocking, 1993; Ferrer et al., 2003); and other factors that
determine the limits of microbial function in hostile envi-
ronments (see Fig. 4; Pitt, 1975; Golyshina et al., 2006;
Hallsworth et al., 2007; Marris, 2008). The current study
illustrates how hitherto unidentified stress parameters can
limit microbial cell function under certain environmental
conditions, and may thereby constrain the biosphere in
specific locations. Further work is needed to identify and
characterize the stress mechanisms that act as failure
points for ecosystems in hostile environments (Fig. 4).
Many informative studies of the geochemical composition
of extreme environments have already been carried out,
including those of other planets (Mustard et al., 2008).
Although chaotropicity has been shown to limit the func-
tional biosphere in specific locations on Earth (Hallsworth
et al., 2007), this stress parameter has not yet been fac-
tored into the mathematical models used to predict the
feasibility of life in as-yet-unexplored environments on
Earth or other planetary bodies (Beaty and Buxbaum,
2006; Marion and Kargel, 2008; Mustard et al., 2008;
Tosca et al., 2008). We believe that chaotropicity should
be accounted for in future models that aim to predict what
types of environment can potentially support cellular
activity (Fig. 4).
Global climate change, and changes in land-use, have
accelerated the expansion of biologically hostile arid and
semiarid regions over the past 30 years (Thomas et al.,
2005; Seager et al., 2007). Polluted environments also
represent a challenge to microbes that are exposed to the
chaotropic activities of xenobiotics (Hallsworth et al.,
2003a). Microbes in other natural habitats, as well as
substrates used in industrial processes, may also be sub-
jected to low water-activity conditions and/or high concen-
microbial
cell function
in hostile
environments
characterization
identification
of components/
processes that
fail under
extreme
conditions ‡
creation of
models to predict
the biological perm -
issivity of diverse
environments §
studies of
macromolecule
structure & function
under extreme
conditions
roles of
compatible solutes,
EPS & other
cellular responses
in stress
tolerance
responses
application
of metagenomic
techniques to
determine metabolic
activity in situ
does not
limit life at low
water activity
chaotropicity
can determine
cellular function
at low water
availability
evidence for
a new class of
extremophilic
microbe that is
chaophilic or
chaotolerant
absolute barriers
to life: cell function
is determined by
the net effect of
environmental
factors
kosmotropic
chaotropic activity
of cellular stressors
hitherto unidentified
‘mining ’ of
chaotrope -
tolerant microbes
for biotechnologically
useful products
microbial
cell function
in hostile
environments
characterization
of solute activities
that constrain
the functional
biosphereb
identification
of components
that fail under
extreme
conditionsc
creation of
models to predict
the biological perm-
issivity of diverse
environmentse
studies of
macromolecule
structure & function
at extreme
temperature &
water activity
roles of
compatible solutes,
EPS & other
cellular responses
in stress
tolerance
characterization
of osmotic, matric &
chaotrope stress &
stress responsesa
application
of metagenomic
techniques to
determine metabolic
activity in situd
per se does not
water activi ty
always limit life at
low water-activity
chaotropicity
can determine
microbial growth
windows at low
water-activity
evidence for
a new class of
extremophilic
microbe that is
chaophilic or
chaotolerant
physicochemical
barriers to cell function
are not absolute;
they are determined by
the net effect of
environmental
factors
enhanced
cellular function by
modifying non-covalent
interactions in &
between cellular
macromolecules
kosmotropic
solutes can offset the
inclusion of
as a stress parameter
function
elucidation
of the stress
mechanisms of
diverse classes
substances
of chaotropic
use of
systems biology
approaches to
understand cell
function in relation
to factors that affect
macromolecular
interactions
interactions
identification
of failure -points
for ecosystem
development in
water-constrained &
other hostile
environments
identification of
stress parameters
& stress
mechanisms
utilization of
macromolecular
systems to quantify
chao-/kosmotropic
activity of complex
natural substrates
chaotropic environments
for hitherto undiscovered
microbes & their
communities
chaotropic activity
predict biosphere
in models to
investigations
of low-solute &
manipulation of
environmental conditions
conditions & utilization
of phenotypic plasticity to
enhance microbial activity
in hostile environ-
ments
Fig. 4. Representation of scientific progress towards understanding the limits of microbial function in hostile environments in relation to
earlier studies (blue), the current study (red), and further studies that are needed (yellow); a. Dutrochet (1826); Griffin (1977); Hallsworth and
colleagues (2003a); b. Scott (1957); Hallsworth and colleagues (2007); c. Hocking (1993); Ferrer and colleagues (2003); d. e.g. Hallsworth
and colleagues (2007); and e. Marion and Kargel (2008).
Limits of life in hostile environments 3303
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology,11, 3292–3308
trations of chaotropic stressors such as formamide,
ethanol, urea, ethylene glycol, butanol, NH4NO3, glycerol,
phenol, MgCl2, CaCl2and sodium benzoate (Brown, 1990;
Hallsworth, 1998; 2003a; 2007; Bardavid et al., 2008). For
both low water-activity and highly chaotropic substrates,
analysis of the ways in which these (and other) stress
parameters interact to limit biological activity can shed
light on how to optimize or eliminate microbial activity, as
required. Chaotropes and high temperatures disorder cel-
lular structures, whereas kosmotropes and low tempera-
tures have a stabilizing/ordering effect (Hamaguchi
and Geiduschek, 1962; Collins, 1997; Hallsworth et al.,
2003a) and we utilized the counteracting solute activities
of glycerol and kosmotropic substances in order to extend
the biotic window for xerophile growth at low water activity
(Fig. 2A–I; Table 4). It may be that microbial cells in labo-
ratory culture could, and that cells in nature do, function
below the water-activity limit of 0.647 established in the
present study (Fig. 2), and that we have not yet under-
stood how to manipulate solute activities sufficiently well
to maintain macromolecular function under high-solute
conditions. Furthermore, the vast majority of microbes
cannot be cultivated in vitro (Whitman et al., 1998; Rutz
and Kieft, 2004; Ward and Fraser, 2005) and some of
these may remain elusive as long as the physicochemical
parameters that determine their biotic windows for growth
are poorly understood. We propose that manipulation of
solute activities will facilitate the cultivation and study of
numerous microbial species that can currently only be
detected in situ (using metagenomic techniques).
Knowledge-based approaches to manipulating environ-
mental conditions could lead to strategies for the regen-
eration of desertified regions (see Kashangura et al.,
2006), and for providing both food and biofuels to support
human population whilst maintaining a sustainable bio-
sphere. Diverse approaches based on manipulation of
environmental conditions or stress parameters have
already given rise to quantum improvements in the growth
windows of mesophilic species (see Hallsworth and
Magan, 1994b; 1995; Thomas et al., 1994; Hallsworth
et al., 2007); there is evidence that kosmotropic sub-
stances reduce ethanol stress in yeast (see Hallsworth,
1998), and that kosmotropic ions can increase Halobac-
terium activity under chaotropic conditions (see Oren,
1983; Hallsworth et al., 2007). The bioremediation of
chaotrope-polluted soils (Hallsworth et al., 2003a) may be
most efficient at temperatures low enough to minimize the
chaotrope-induced disordering of cellular structures. The
effectiveness and efficiency of products and processes
such as biocides, food preservatives (e.g. sodium ben-
zoate), food and drinks fermentations, bioalcohol pro-
duction from microbes (Hallsworth, 1998), and industrial
biocatalysis in solvent systems, which utilize or generate
chaotropic solutes could be enhanced via manipulation of
solute activities. Further studies are needed so that more
effective interventions can be made based on exploitation
of phenotypic plasticity, employing recombinant technolo-
gies and/or systems biology approaches to obtaining
stress-resistant cells (see Fig. 4).
Xerophilic fungi have most commonly been isolated
from kosmotrope-containing substrates (Pitt, 1975), so
chaophilic microbes in nature may have thus far gone
unnoticed. Further, high-solute habitats have typically
been the focus of searches for xerophilic microbes so
countless xerophilic species (and indeed whole commu-
nities) on low-solute surfaces may have been overlooked.
It is intriguing to speculate what proportion of xerophilic
fungi in nature are also chaophilic, and what proportion
are restricted to either high-solute or low-solute sub-
strates. Furthermore, extremophilic fungi already act as
important biotechnological resource (Archer and Peberdy,
1997), and chaotolerant species and/or their enzymes
may have potential for diverse applications. One focus of
our ongoing studies is to identify novel stress parameters
that prevent life processes: it may be possible to further
enhance microbial function, and ecosystem development,
in hostile environments once the stress biology of
microbes has been more completely elucidated.
Experimental procedures
Sampling strategies and environmental isolates
Fungi were isolated from diverse environments (see Table 1)
using sterile cotton-tip swabs and inoculated onto slants of
Malt-Extract, Yeast-Extract Phosphate Agar [MYPiA; 1%
Malt-Extract w/v (Oxoid, UK), 1% Yeast-Extract w/v (Oxoid,
UK), 1.5% Agar w/v (Acros, USA), 0.1% K2HPO4w/v] supple-
mented with 5 M glycerol (0.845 aw) in 1.8 ml, internal-thread
cryovials and transferred, once back in the laboratory, to Petri
plates containing MYPiA supplemented with glycerol (5 M;
0.845 aw) or sucrose (2.2 M; 0.884 aw). Petri plates were
incubated at temperatures between 20°C and 30°C in sealed
bags (see below) and checked periodically for up to
6 months. Upon visual inspection, all isolates that had grown
were subcultured onto MYPiA supplemented with 6.52 M
glycerol, and incubated at 30°C.
Named xerophile species
Named xerophile species were obtained from Culture Collec-
tion of Yeasts (CCY, Bratislava, Slovakia), German Collection
of Microorganisms and Cell Cultures (DSMZ, Braunschweig,
Germany), Extremophilic Fungi Culture Collection (EXF,
Ljubljana, Slovakia), Food Research Ryde (FRR, North
Ryde, Australia), International Mycological Institute (IMI,
Egham, UK), and University of Western Ontario, UWOPS
Collection (Ontario, Canada); see Table 2. Cultures were
maintained on MYPiA supplemented with either 2.78 M
glucose (0.911 aw) or 2.2 M sucrose (0.884 aw) in sealed bags
(see below) and incubated at 30°C.
3304 J. P. Williams and J. E. Hallsworth
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology,11, 3292–3308
Culture conditions and growth-rate determinations
Throughout this study, all media were sterilized in Schott
bottles in a water bath (100°C, 60 min), cooled to within 7.5°C
of the medium gel-point, and then poured into 9 cm vented
Petri plates; inoculations were carried out using 4 mm diam-
eter plug from the periphery of actively growing cultures
growing on MYPiA media supplemented with 5.43 M glycerol;
and plates containing identical media were sealed in polyeth-
ylene bags to maintain a constant water activity (see
Hallsworth et al., 2003b). Growth was assessed at periodic
intervals by taking two measurements of colony diameter (in
perpendicular directions), that were used to calculate rates of
radial extension as described previously (Pitt and Hocking,
1977; Hallsworth and Magan, 1994a). Two-dimensional
stress-tolerance profiles were plotted as described previously
(Hallsworth and Magan, 1999) and mean values were plotted,
and variation indicated in each display. Growth of yeast was
carried out on the same media and quantified using spot tests
as detailed by Albertyn and colleagues (1994).
Modification of media to investigate stress tolerance
Single-stressor solute-tolerance screen. All environmental
isolates and named fungal strains were screened for stress
tolerance on MYPiA media supplemented with different
solutes over a range of concentrations; either ethanol
(0.4–1.3 M), NaCl (2.1–3.4 M), ethylene glycol (2.0–3.4 M),
glycerol (2.5–5.0 M), MgCl2(0.7–1.5 M), fructose (3.0–
4.8 M), sucrose (1.1–2.1 M), or polyethylene glycol (PEG)
400 (0.8–1.3 M), without addition of pH buffer, and incubated
at 30°C for a period of up to 90 days. All inoculations were
carried out in triplicate.
pH-tolerance study. All environmental isolates and named
fungal strains were grown on MYPiA media supplemented
with glycerol (at 3.8 M; 0.92 awand 4.4 M; 0.88 awrespec-
tively) and buffered to pH values of: 3.75, 4.5, 5.75 (citric
acid; Na2HPO4), 6, 6.75 (MES; NaOH) and 7.5 (Hepes;
NaOH; see Hallsworth and Magan, 1996). The pH of each
medium was adjusted prior to autoclaving using appropriate
buffers then measured postautoclave using a Mettler Toledo
Seven Easy, pH-probe (Switzerland).
Temperature: water-activity growth-response study. The nine
most xerophilic strains were inoculated onto MYPiA media
supplemented with four different concentrations of glycerol
(5.43, 6.19, 6.84 and 7.44 M) with water activity values
ranging from 0.81 and 0.65 (see Fig. 1C–K) and incubated at
15, 22.5, 30 and 37.5°C. Quantification of water activity is
described below.
Mixed-solute media for limits-of-cell function at low water-
activity study. The nine xerophilic strains were inoculated
onto 14 ultra-low water-activity media (0.714–0.644 aw), con-
sisting of MYPiA media supplemented with combinations of
glycerol, fructose, or glycerol plus kosmotropic solutes (see
Fig. 2, Table 3, Fig. S1). The pH of all media was adjusted to
5.75, unless stated otherwise, using citric acid: Na2HPO4
buffer; following inoculation Petri plates were incubated at
30°C. For water-activity and chaotropic-activity values of
Media 1–14 see Table 3; the methodologies used to obtain
these values are described below.
Quantification of water activity
The water-activity values of media were measured at 30°C or,
if different, at the temperature of incubation using a Novasina
IC-II water-activity machine fitted with an alcohol-resistant
humidity sensor and eVALC alcohol filter (Novasina,
Pfäffikon, Switzerland), as described previously (Hallsworth
and Nomura, 1999). This equipment was calibrated using
saturated salt solutions of known water activity (Winston and
Bates, 1960). Values were determined three times using rep-
licate solutions made up on separate occasions. The varia-
tion of replicate values was within ⫾0.002 aw.
Determination of chaotropic activity
The chaotropic activity of solute(s) used to supplement
growth media (see Table 3) was measured as a function of
their ability to destabilize the polysaccharide macromolecule
agar (Extra-Pure Reagent-grade agar, gel strength 600–
700gcm
-2, from Nacalai Tesque, Kyoto, Japan), and thereby
lower gel-point (Hallsworth et al., 2003a). Agar was melted in
distilled water, cooled to 55°C, and added to a solution of the
solute or solute-mixture to be tested, also at 55°C, to give a
final concentration of agar of 1.5% w/v and concentration(s)
of solute(s) as used for the growth study. The agar–
compound solutions were allowed to cool gradually and the
gel-point temperature (⫾0.3°C) was recorded using a tem-
perature probe (Jenway, UK). The gel points determined
were used to calculate the chaotropic activity of each com-
pound in kJ kg-1(mole added compound)-1, based on the
fact that the heat capacity for a 1.5% agar w/v gel is
4.15 kJ kg-1°C-1(see Hallsworth et al., 2003a).
Acknowledgements
We are grateful for scientific discussions with A.N. Bell, P.
Bhaganna, A.G. Maule, J.P. Quinn, D.J. Timson (Queen’s
University Belfast, Northern Ireland), K.D. Collins (University
of Maryland, USA), M.J. Danson (University of Bath, UK), J.L.
Finney (University College London, UK), F. Franks (BioUp-
date Foundation, UK), E.A. Galinski (University of Bonn,
Germany), A.D. Hocking (CSIRO Division of Food Science
and Technology,Australia), T.J McGenity and P. Nicholls (Uni-
versity of Essex, UK), A.Y. Mswaka (University of Harare,
Zimbabwe), R.P. Rand (Brock University, Canada), R.J.P.R.
dos Santos (Laboratório de Análises of the Instituto Superior
Técnico, Portugal), and K.N. Timmis (Helmholtz Centre for
Infection Research, Braunschweig, Germany); and also the
Reviewers of the manuscript who offered new insights into
the data. We wish to thank N. Gunde-Cimerman (University
of Ljubljana, Slovenia), M.-A. Lachance, University of
Western Ontario, Canada) and R.J.P.R. dos Santos (Portu-
gal) for providing strains of yeast and fungi. Funding was
received from the Department of Education and Learning
(Northern Ireland), the Great Britain Sasakawa Foundation
(London, UK), the Natural Environment Research Council
Limits of life in hostile environments 3305
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology,11, 3292–3308
(UK; Grant No. NEE0168041), the Biotechnology and
Biological Sciences Research Council (UK; Grant No.
BBF0034711), Queen’s University Belfast Promising
Researcher Fund and the MacQuitty Charitable Foundation
(Queen’s University Belfast).
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Supporting information
Additional Supporting Information may be found in the online
version of this article:
Fig. S1. Flow-chart to illustrate the progression of research
activities during the current study.
Table S1. Fungal species capable of germination ⱕ0.71
water activity.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
3308 J. P. Williams and J. E. Hallsworth
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