life
Communication
A New Record for Microbial Perchlorate Tolerance:
Fungal Growth in NaClO4Brines and its Implications
for Putative Life on Mars
Jacob Heinz 1, Tim Krahn 1and Dirk Schulze-Makuch 1,2,3,4,*
1
Astrobiology Research Group, Center for Astronomy and Astrophysics (ZAA), Technische Universität Berlin,
2Section Geomicrobiology, German Research Centre for Geosciences (GFZ), 14473 Potsdam, Germany
3
Department of Experimental Limnology, Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB),
12587 Stechlin, Germany
4School of the Environment, Washington State University, Pullman, WA 99163, USA
*Correspondence: [email protected]
Received: 14 April 2020; Accepted: 24 April 2020; Published: 28 April 2020
Abstract:
The habitability of Mars is strongly dependent on the availability of liquid water, which is
essential for life as we know it. One of the few places where liquid water might be found on Mars is
in liquid perchlorate brines that could form via deliquescence. As these concentrated perchlorate
salt solutions do not occur on Earth as natural environments, it is necessary to investigate in lab
experiments the potential of these brines to serve as a microbial habitat. Here, we report on the sodium
perchlorate (NaClO
4
) tolerances for the halotolerant yeast Debaryomyces hansenii and the filamentous
fungus Purpureocillium lilacinum. Microbial growth was determined visually, microscopically and
via counting colony forming units (CFU). With the observed growth of D. hansenii in liquid growth
medium containing 2.4 M NaClO
4
, we found by far the highest microbial perchlorate tolerance
reported to date, more than twice as high as the record reported prior (for the bacterium Planococcus
halocryophilus). It is plausible to assume that putative Martian microbes could adapt to even higher
perchlorate concentrations due to their long exposure to these environments occurring naturally on
Mars, which also increases the likelihood of microbial life thriving in the Martian brines.
Keywords:
Mars; habitability; brines; salts; perchlorate; microorganisms; halotolerant; fungi; yeast;
microbial growth
1. Introduction
Several observations on Mars, such as characteristic surface morphologies like large fluid-eroded
channels, dendritic networks, fluvial valleys, and glacial features [
1
], and the formation of
water-depending minerals like hematite [
2
], indicate that the planet had a warmer (even though mostly
freezing [
3
]), wetter and more habitable climate in its early history [
4
]. However, the loss of its magnetic
field enabled the solar wind to sputter away large parts of the Martian atmosphere which caused a
climate change leading to the dry, cold, and hostile planet that we know today [
5
,
6
]. Hence, putative
Martian microorganisms would have had to adapt to a gradual decrease in the availability of liquid
water. In this process of decreasing water activities, one of the last niches for the occurrence of liquid
water would be deposits of hygroscopic salts that absorb water from the thin Martian atmosphere [
7
].
If these salts can absorb enough water, they will dissolve in the absorbed liquid and form a saturated
salt solution (called “brine”) that can be diluted by further water absorption. This process is called
deliquescence. Indeed, there is strong evidence for the occurrence of deliquescence processes and of at
least temporarily stable brines on Mars [8–10].
Life 2020,10, 53; doi:10.3390/life10050053 www.mdpi.com/journal/life
Life 2020,10, 53 2 of 8
Several very hygroscopic salts have been detected on Mars that could undergo deliquescence
processes [
11
]. Among these, the most hygroscopic class of salts are perchlorates which have been found
in the Martian soil at the Phoenix landing site at concentrations of 0.4–0.6 wt.% [
12
]. Perchlorate brines
have very low freezing points, down to
−
77.5
◦
C for a calcium perchlorate solution with an eutectic
concentration [
13
], i.e., the salt concentration causing the most intense freezing point depression. This
also enables the provision of liquid water at subzero temperatures and, hence, provides a promising
cryo-environment that might serve as a habitat for putative Martian microbes.
On Earth, most of the salt-rich habitats are based on sodium chloride (NaCl), e.g., in the Atacama
Desert, Chile, where endolithic cyanobacteria thrive in NaCl crusts by gaining water absorbed by
the salt [
14
,
15
]. Furthermore, it has also been shown very recently that methanogenic archaea can
survive saturated NaCl concentrations and use water exclusively provided by the deliquescence of
salt [
16
]. There are several environments on Earth that also provide high concentrations of salts other
than NaCl, e.g., the Dead Sea with increased calcium (0.47 M Ca
2+
) and magnesium (1.98 M Mg
2+
)
chloride concentrations (additional to saturated NaCl conditions) [
17
], the Spotted Lake (Canada)
containing high sulfate concentrations (>3 M) [
18
], the Don Juan Pond (Antarctica) containing 3.7 M
CaCl
2
[
19
,
20
], or the Discovery Basin (Mediterranean Sea) containing 5 M MgCl
2
[
21
]. While the Dead
Sea and the Spotted Lake are inhabited by halophilic microorganisms [
18
,
22
], the Don Juan Pond and
the Discovery Basin are thought to be sterile environments due to low water activities, high ionic
strengths and chaotropic stress [21,23,24].
In contrast to Mars, there are only very few natural environments on Earth where low perchlorate
concentrations have been detected, e.g., in the Atacama Desert, Chile [
25
–
27
], and in the Dry Valleys,
Antarctica [
28
], and no environment is known that consists of concentrated perchlorate brines. Thus,
it is appropriate to study these brines in lab experiments to better understand their potential in
serving as a habitat for microbial life on Mars. To date, there is only a relatively small body of
literature investigating the habitability of brines with perchlorate concentrations above ~ 0.1 M [
29
–
38
].
More studies exist on perchlorate-reducing bacteria (for reviews see [
39
,
40
]) and archaea [
41
] at low
perchlorate concentrations below 0.1 M. Prior to this study, the organism found tolerating the highest
perchlorate concentration suitable for microbial growth was the halotolerant bacterial strain Planococcus
halocryophilus, which tolerates 1.1 M sodium perchlorate (NaClO
4
) in its liquid growth medium at
25
◦
C [
30
]. Earlier studies reported lower perchlorate tolerances for halotolerant bacteria and halophilic
archaea (see Table 1). However, there have been no investigations on the perchlorate tolerance of
eukaryotes prior to this study, even though fungi are known to tolerate high concentrations of various
other salts [42,43].
Table 1.
Sodium perchlorate (NaClO
4
) tolerances (in mol/L, wt.% [w/w], and wt./vol.% [w/v]) for the
two organisms of each domain of life tolerating the highest perchlorate concentrations reported to date.
Domain Organism NaClO4Tolerance Literature
(mol/L) (wt.%) (wt./vol.%)
Archaea Haloferax mediterranei 0.6 6.8 7.3 [31]
Halorubrum lacusprofundi 0.8 8.9 9.8 [32]
Bacteria Halomonas venusta 1.0 10.9 12.2 [33]
Planococcus halocryophilus
1.1 12.0 13.6 [30]
Eukarya
(Fungi)
Purpureocillium lilacinum 1.9 19.0 23.5 This study
Debaryomyces hansenii 2.4 23.0 29.9 This study
Here, we report on the NaClO
4
tolerance of the halotolerant yeast Debaryomyces hansenii and the
filamentous fungus Purpureocillium lilacinum.D. hansenii can be found in hyper-saline environments
like the Great Salt Lake of Utah or in salterns on the Atlantic coast of Namibia [
44
]. The yeast has been
shown to grow in media containing up to 4 M NaCl [
44
]. Its high halotolerance results mainly from the
accumulation of the compatible solutes glycerol and arabinitol in the exponential growth phase and
Life 2020,10, 53 3 of 8
in the stationary phase, respectively [
45
]. Additional changes in the metabolism of the yeast during
growth under saline conditions are reviewed elsewhere [
44
,
46
,
47
]. In contrast, there is no detailed
research on the halotolerance of P. lilacinum. Lotlikar and Damare (2018) [
48
] showed that the fungus
can grow in a medium with a salinity of S =100 (corresponding to a NaCl concentration of 1.9 mol/L),
but not at a salinity of S =250 (5.7 mol/L NaCl). Arpini et al. (2019) [
49
] found the minimum inhibitory
concentration (MIC) for NaCl to be 200 g/L (3.4 mol/L NaCl) for P. lilacinum.
2. Materials and Methods
2.1. Organisms and Culture Conditions
The halotolerant yeast Debaryomyces hansenii (DSM 3428) was obtained from the DSMZ (Leibniz
Institute DSMZ—German Collection of Microorganisms and Cell Cultures). The yeast cells were
grown aerobically at 25
◦
C (optimum growth temperature) in liquid DMSZ growth medium #90 (3%
malt extract, 0.3% soya peptone) with various concentrations of NaClO
4
. The media were prepared
by mixing the media components, NaClO
4
and water, followed by pH adjustment (pH ~5.6) and
sterile filtration.
The filamentous fungus Purpureocillium lilacinum was found as a contamination at surprisingly
high NaClO4concentrations (see Results) during growth experiments with Planococcus halocryophilus
in liquid DMSZ growth medium #92 (3% tryptic soy broth, 0.3% yeast extract, pH 7.2–7.4, 25
◦
C)
which have been described elsewhere [
30
]. P. lilacinum was isolated and characterized by 18S rDNA
sequencing (data not shown). Due to safety restrictions (P. lilacinum is categorized as biosafety level S2)
no further experiments were conducted with this fungus.
2.2. Determination of Perchlorate Tolerances
The survival and growth of the fungi were determined visibly in 15 mL centrifuge tubes containing
the inoculated liquid growth medium, as well as by using light microscopy (Primo Star, Zeiss, equipped
with Axio Cam 105 color) and counting colony forming units (CFU) on agar plates (1.5% agar)
containing the respective growth medium. The maximum NaClO
4
concentration suitable for growth
was determined through progressive culture adaptation to higher perchlorate concentrations as
described previously [
30
]. In short, 10
µ
l of a stock culture was used to inoculate 5 ml of liquid growth
medium containing 10 wt.% NaClO
4
(corresponding to 0.9 mol/L). The culture growing from this
medium was used to inoculate growth medium containing 15 wt.% NaClO
4
(1.4 mol/L). This procedure
was repeated with increasing NaClO
4
concentrations in 5 wt.% steps. When no growth could be
detected the increments of the NaClO
4
concentration increase were lowered to 1 wt.%. The highest
NaClO
4
concentration that enabled growth was defined as the perchlorate tolerance of the organism
with a technical error of
±
1 wt.% NaClO
4
. All growth experiments were conducted as biological
duplicates, i.e., for each NaClO4concentration, two separate samples were inoculated.
3. Results
The perchlorate tolerance of D. hansenii was found to be 2.4 M NaClO
4
. Growth curves (until the
exponential growth phase) for the samples with this concentration are shown in Figure 1together with
the curves for samples with a concentration of 2.6 M NaClO
4
, in which the cells were dying within
10 days. Additionally, inserted into Figure 1is a light microscopy image of D. hansenii cells after growth
in DSMZ medium #90 showing single cells and some small and loosely-bound cell aggregates. Besides
a decrease in cell density, no phenotypical changes in the yeast cell morphologies were found when the
cells were grown in perchlorate-rich media. This contrasts with earlier studies on the bacterial strain
P. halocryophilus where cells grown in perchlorate-rich media formed large cell clusters [30].
Life 2020,10, 53 4 of 8
Processes 2020, 8, x; doi: FOR PEER REVIEW www.mdpi.com/journal/processes
Figure 1.
Growth curves of D. hansenii at 25
◦
C in liquid growth media with a sodium perchlorate
(NaClO
4
) concentration of 2.4 M (green line and symbols) and 2.6 M (red line and symbols). Experiments
were run in biological duplicates (samples A and B). Microbial growth occurred only in the 2.4 M
NaClO
4
samples while the cells in the 2.6 M NaClO
4
samples died within 10 days. Cells of D. hansenii
after growth in liquid growth medium are shown in the implemented image.
Purpureocillium lilacinum was not investigated in detail due to safety restrictions (see Section 2.1).
However, it was found as a contaminant growing in liquid growth medium having NaClO
4
concentrations of up to 1.9 M, which we interpret to be its uppermost tolerance to NaClO4.
4. Discussion
Before the detection of significant amounts of perchlorate on Mars in 2008 [
12
], the scientific
interest in determining the habitability of concentrated perchlorate brines was low due to their practical
non-existence in natural habitats on Earth. Since 2008, the number of studies investigating this
question has increased but is still insufficient for understanding the potential for life in Martian
perchlorate-containing brines. Table 1(see Introduction) lists the two record holders for each domain
of life regarding their NaClO4tolerance as described in the literature thus far.
The study presented here is the first one describing significant perchlorate tolerances for eukaryotes.
Several other studies described growth of non-fungal organisms in perchlorate solutions with
concentrations lower than the ones listed in Table 1[
31
,
33
–
38
]. For example, Oren et al. (2014) [
31
]
found that several halophilic archaea of the family Halobacteriaceae (Halobacterium strain NRC-1,
Hbt. salinarum R1, Haloferax volcanii,Hfx. mediterranei,Hfx. denitrificans,Hfx. gibbonsii,Haloarcula
marismortui, and Har. vallismortis) and the bacterium Halomonas elongata grew well in NaCl-based
media supplemented with a perchlorate concentration of up to 0.4 M. However, among these, only
Hfx. mediterranei was also able to grow in 0.6 M NaClO
4
. Al Soudi et al. (2017) [
33
] found that the
halotolerant bacterial strains Marinococcus halophilus,Halomonas venusta, and Bacillus licheniformis grew
Life 2020,10, 53 5 of 8
robustly at 0.5 M NaClO
4
, while only H. venusta also showed substantial growth at 1.0 M NaClO
4
.
Furthermore, it has been shown that methanogenic archaea can metabolize and produce methane at
perchlorate concentrations of up to 0.4 mol/L [38].
Therefore, the two fungi investigated in this study, D. hansenii and P. lilacinum, have by far
the highest tolerances to NaClO
4
among all microorganisms investigated to date. The tolerance for
D. hansenii (2.4 M NaClO
4
) is more than twice as high as for the bacterial strain P. halocryophilus that has
been holding the NaClO
4
tolerance record (1.1 M NaClO
4
) prior to this study [
30
]. As the two fungi
described in this study are the first ones ever investigated regarding their perchlorate tolerance, it is
plausible to assume that other fungi (e.g., the extremely halotolerant black yeast Hortaea werneckii, or the
obligately halophilic Wallemia ichthyophaga [
43
]) might tolerate even higher perchlorate concentrations,
which we plan to investigate in upcoming experiments.
The perchlorate tolerance data available in the literature to date (Table 1) convey the impression
that fungal species have a more efficient perchlorate defense machinery than bacteria and archaea.
However, more research on microbial and fungal perchlorate tolerances and the adaptation mechanisms
applied by the fungi grown in perchlorate-rich growth media is needed to confirm or discard this
hypothesis. Furthermore, the effect of additional stress factors typical for Mars on the fungal survival
and growth in perchlorate brines should prompt further investigations. The most relevant stressors
in this context are low temperatures and pressures, high radiation levels, and stress induced by the
higher chaotropicity and ionic strengths of ions from other perchlorate salts like magnesium or calcium
perchlorate, which probably represent the majority of perchlorate salts on Mars [50,51].
Since there are no natural perchlorate-rich environments existing on Earth and thought not to have
existed in the past, there is no obvious adaptation mechanism for the two fungal species investigated
in this study to have adapted to these high NaClO
4
concentrations. If life on Mars exists in perchlorate
brines, we may speculate that these microorganisms might have evolved tolerance to much higher
perchlorate concentrations, as they—in contrast to Earth—would have been under natural selection
pressures on Mars to achieve higher perchlorate tolerances. We recommend that this hypothesis
should be tested in future Mars missions via life detection experiments in saline and perchlorate-rich
environments, such as locations where the presumably deliquescence-driven “recurring slope lineae”
(RSL) [10] occur.
Author Contributions:
Conceptualization, J.H. and D.S.-M.; methodology, J.H.; validation, J.H., T.K. and D.S.-M.;
formal analysis, J.H. and T.K.; investigation, J.H. and T.K.; resources, D.S.-M.; writing—original draft preparation,
J.H.; writing—review and editing, J.H., T.K. and D.S.-M.; supervision, D.S.-M.; project administration, D.S.-M.;
funding acquisition, D.S.-M. All authors have read and agreed to the published version of the manuscript.
Funding:
Research was funded by the European Research Council Advanced Grant “Habitability of Martian
Environments” (HOME, no. 339231).
Acknowledgments:
We thank Hans-Peter Grossart and Solvig Pinnow for conducting the 18S rDNA sequencing
of Purpureocillium lilacinum and two anonyms reviewers for helping us to improve this article.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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