Recent evidence from the Opportunity and Spirit rovers and the Mars Express mission suggests that the soils on Mars might be very high in biotoxic materials including sulfate salts, chlorides, and acidifying agents. Yet, very little is known about how the chemistries of Mars soils might affect the survival and growth of terrestrial microorganisms. The primary objectives of the research included: (1) prepare and characterize Mars analog soils amended with potential biotoxic levels of sulfates, chlorides, and acidifying minerals; and (2) use the simulants to conduct a series of toxicology assays to determine if terrestrial microorganisms from spacecraft can survive direct exposure to the biotoxic soils

Golden, D. C.

Ming, Doutlas W.

Schuerger, A. C.

NASA Technical Reports Server

BIOTOXICITY OF MARS ANALOG SOILS: MICROBIAL DISPERSAL INTO DESICCATED SOILS 
VERSUS EMPLACEMENT IN SALT OR ICE INCLUSION FLUIDS.  A. C. Schuerger1, D. W. Ming2, and D. 
C. Golden2.  1University of Florida, Bldg. M6-1025, Space Life Sciences Lab, Kennedy Space Center, FL 32899; 
email: schuerg@ufl.edu; 2Astromaterials Research and Exploration Science Office, Mail Code JE23, NASA JSC, 
Houston, TX 77058; emails douglas.w.ming@nasa.gov and d.c.golden1@nasa.gov, respectively.   
 
Introduction: Recent evidence from the Opportunity 
and Spirit rovers and the Mars Express mission suggests that 
the soils on Mars might be very high in biotoxic materials 
including sulfate salts, chlorides, and acidifying agents [1,2]. 
Yet, very little is known about how the chemistries of Mars 
soils might affect the survival and growth of terrestrial mi-
croorganisms. 
In a recent paper on the interactive effects of hypobaria, 
low temperatures, and CO2-enriched atmospheres on the 
growth of seven Bacillus spp., Schuerger and Nicholson [3] 
identified 13 potential biocidal agents that might affect mi-
crobial survival and growth on the martian surface.  These 
factors include the following (not in priority): (1) solar UV 
irradiation, (2) low pressure, (3) extreme desiccating condi-
tions, (4) extreme diurnal temperature fluctuations, (5) solar 
particle events, (6) galactic cosmic rays, (7) UV-glow dis-
charge from blowing dust, (8) solar UV-induced volatile 
oxidants [e.g., O2–, O–, H2O2, O3], (9) globally distributed 
oxidizing soils, (10) extremely high salts levels [e.g., MgCl2, 
NaCl, FeSO4, and MgSO4] in surficial soils at some sites on 
Mars, (11) high concentrations of heavy metals in martian 
soils, (12) likely acidic conditions in martian fines, and (13) 
high CO2 concentrations in the global atmosphere.  The 
Phoenix mission’s discovery of perchlorates in the polar 
regolith [4] adds a 14th factor to this list.  Despite these ex-
treme conditions many studies have demonstrated that spores 
or dormant cells of terrestrial microorganisms can survive 
simulated conditions on Mars as long as they are protected 
from solar UV irradiation [5,6,7,8].  What has not been ex-
plored in depth has been the effects of potential biotoxic 
components in the martian regolith on the survival, growth, 
and adaptation of terrestrial microorganisms on Mars.   
Nature of the martian regolith.  The fine-grained surfi-
cial dust or regolith on Mars is often referred to in the litera-
ture as the “martian soil” [9].  The “global” soil is highly 
oxidized and contains basaltic materials (possibly from local 
basaltic rocks), nano-phase iron oxides, and SO4  and Cl  
bearing salts [1].  The chemistry of the fine-grained regolith 
or “soil” at the Pathfinder site, the two Viking landing sites, 
and the MER Spirit and Opportunity sites site are very similar, 
which is attributed to global mixing of fine-grained materials 
due to winds [9,10,11].  Minor variations in regolith composi-
tions, especially for K2O and Na2O, have been observed in 
soil compositions at Pathfinder, Viking, and MER sites.  
Orbital gamma ray data also suggest that regional variations 
in K+ and Cl  are present [12].  The new MER data confirms 
that the chemically uniform fine-grained regolith probably 
represents a major surface component on Mars [10].  How-
ever, there are soils that have very high concentrations of 
sulfate salts (e.g., Fe-, Mg-, and Ca-sulfates in the soil called 
“Paso Robles” in Gusev crater) [1]. 
The primary objectives of the research included: (1) pre-
pare and characterize Mars analog soils amended with poten-
tial biotoxic levels of sulfates, chlorides, and acidifying min-
erals; and (2) use the simulants to conduct a series of toxi-
cology assays to determine if terrestrial microorganisms from 
spacecraft can survive direct exposure to the biotoxic soils.   
Materials and Methods:  A Mars Simulation 
Chamber (MSC) was used to recreate conditions simi-
lar to equatorial Mars (described in full by Schuerger 
et al. [13].  The MSC system can accurately simulate five 
key components of the surface environment of Mars includ-
ing: (a) pressures down to 0.1 mb, (b) UVC, UVB, and UVA 
irradiation from 190 to 400 nm, (c) dust loading in the at-
mosphere from optical depths of 0.1 to 3.5, (d) temperatures 
from –100 to +30 C, and (e) atmospheric mix composed of 
the top five gases in the martian atmosphere [CO2 (95.53%), 
N2 (2.7%), Ar (1.6%), O2 (0.13%) and H2O (0.03%)].  
Two toxicological assays have been conducted with cul-
turable mesophilic microbial species typically recovered 
from spacecraft surfaces.  First, spores or vegetative cells of 
individual culturable species were applied to pre-sterilized 
iridited aluminum coupons, dried, covered with Mars analog 
soils to depths of up to 1 cm, placed in the Mars chamber, 
and incubated for 7 d at Mars conditions similar to those 
experienced by the Viking, Pathfinder, Opportunity and 
Spirit missions.  Second, spores or vegetative cells of cultur-
able species were added to water extracts of six Mars analog 
soils to determine if terrestrial microorganisms are capable of 
survival in hypersaline soil solutions.  The soil solutions 
were obtained by vigorously mixing 50 g of each soil in 100 
ml of 18 ohm deionized water for 2 hrs, filtered sequentially 
three times through cellulosic filters of 30, 0.45, and 0.22 µm 
nominal pore sizes, and dispensed into separate glass test 
tubes.  Approximately 2 x 107 cfu’s of spores or vegetative 
cells were added to each tube of soil solutions and then incu-
bated at 24, 0, or  70 C for 7 d. 
The “dry-assay” was designed to simulate bioloads on 
spacecraft surfaces covered by desiccated Mars soils during 
active descent onto the martian surface (i.e., rocket exhaust 
kicking up dusts that settle onto the upper decks of landers).  
The “wet-assay” was designed to simulate microbial species 
being emplaced into ice or salt inclusions during drilling 
operations by a rover.   
Mesophilic species used in the dry-assays included Ba-
cillus subtilis, Enterococcus faecalis, Escherichia coli, Pseu-
https://ntrs.nasa.gov/search.jsp?R=20100005761 2019-08-30T08:54:37+00:00Z
domonas aeruginosa, Serratia liquifaciens, and Staphylococ-
cus aureus.  Species used in the wet-assay included B. sub-
tilis and E. faecalis.  Following exposure to experimental 
conditions, all materials doped with endospores or vegetative 
cells of microorganisms were assayed for viable numbers of 
surviving spores or cells using a Most Probable Numbers 
(MPN) procedure originally described by Mancinelli and 
Klovstad [14] and modified by Schuerger et al., [7,8].   
Six Mars analog soils were prepared to simulate a range 
of potentially biotoxic soils and are derived from Mars Path-
finder, MER, and Phoenix data [1,2,9,10,11,12].  The soils 
include simulants for (a) a ground basalt that served as a non-
toxic control (soil solutions = pH 8.62; EC 59 µS/cm), (b) 
acidic soil (pH 2.0; EC 38.8 mS/cm), (c) high salt soil (pH 
3.14; EC 18.4 mS/cm), (d) alkaline soil (pH 10.18; EC 12.5), 
(e) perchlorate soil (pH 7.73; EC 5.56 mS/cm), and (f) aeo-
lian soil that combined materials from all soils (pH 7.03; EC 
6.71 mS/cm).   
Results for the Dry-Assay: A desiccation experiment 
indicated that endospores of B. subtilis were very resistant to 
drying onto iridited aluminum coupons (used to simulate 
spacecraft surfaces), but that desiccation of all non-spore 
forming species yielded losses of viable cells between one to 
six orders of magnitude.  Vegetative cells of Serratia lique-
faciens were the most sensitive and cells of Enterococcus 
faecalis were the most resistant to desiccation of the non-
spore forming species tested.  A 7-d Mars simulation was 
conducted with endospores of B. subtilis and vegetative cells 
of E. faecalis to measure survival rates of both species when 
covered by 2 mm of either the basalt “control” soil or 2 mm 
of the high-salt soil and exposed to 6.9 mb total pressure,      
 10 C, continuous UVC irradiation at 4 W/m2, and main-
tained within a Mars gas mix [7,13].  Cells of E. faecalis 
covered by the high-salt soil and exposed to martian condi-
tions, with or without UV irradiation, were killed off to be-
low detection limits of the assay within 7 d.  Endospores of 
B. subtilis were reduced 1-2 orders of magnitude.  All con-
trols of both species exhibited high survival rates, and the 
interactive effects of (in order of biocidal activity) high-salt 
Mars analog soil, desiccation, and low pressure were con-
cluded to be responsible for the significant decreases in vi-
able numbers by both species.   
Results for the Wet-Assay: In contrast, a wet-assay was 
conducted with B. subtilis and E. faecalis (as of this writing 
the E. faecalis experiments were still underway) in which 
endospores or vegetative cells, respectively, were exposed to 
aqueous soil solutions derived from all 6 soils listed above.  
Results for B. subtilis indicated no significant differences 
between controls and spores exposed to alkaline, perchlorate, 
or aeolian soil solutions.  A very slight decrease in surviving 
spores was noted for the soil solutions from the high-salt and 
acid soil solutions.  The decrease was only ½ order of magni-
tude below the controls and was close to the accuracy of the 
assay.  Thus, for at least endospores of B. subtilis, the aque-
ous soil extractions did not appear to be biotoxic.  Data for E. 
faecalis will be presented at the conference.   
Discussion:  Similar to several other microbial survival 
studies under martian conditions [5,6,7,8,15], interactive 
effects appear to impart significantly more stress on both 
endospores and vegetative cells than individual factors tested 
alone.  The interactive effects of high-salt soil, desiccation, 
and low-pressure significantly decreased the viable cells 
recovered for both B. subtilis and E. faecalis.  In contrast, 
endospores of B. subtilis exhibited only a slight decrease of 
no more than ½ an order of magnitude when exposed for 7 d 
in soil solutions from high salt and acid soils.  No differences 
were observed for the soil solutions derived from the other 
Mars analog soils.   
Experiments with multiple factors present on Mars that 
are likely to yield strong interactive effects are difficult to 
conduct due to requirements for large sample sizes and com-
plex Mars simulators.  For example, to conduct all possible 
interactions for the 14 biocidal factors (listed above) present 
on Mars would require 8.72 x 1010 possible combinations of 
experimental factors (i.e., 14 factorial).  Something that is 
clearly outside the scope of human scientific study.  How-
ever, because multiple biocidal factors are certainly present 
on Mars, combinations of stressing agents need to be tested if 
the astrobiology community is to gain an accurate assessment 
of how terrestrial microorganisms may, or may not, be able 
to survive, metabolize, replicate, and evolve on the surface of 
Mars.  We recommend the following factors as the top priori-
ties for interactive microbiology studies under simulated 
martian conditions: (1) anaerobic atmospheres, (2) high UVC 
fluence rates, (3) pressures near 6.9 mb, (4) desiccation, (5) 
biotoxicity of martian fines, and (6) soil oxidants.   
References: [1] Ming et al. (2006) JGR, 
111(EO2S12), doi:10.1029/2005JE002560. [2] Gen-
drin et al (2005) Science, 307, 1587-1591. [3] 
Schuerger and Nicholson (2006) Icarus, 185, 143-152. 
[4] Hecht et al. (2009) Science, 325, 64-67. [5] Cockell 
et al. (2001) Astrobiology, 5, 127-140. [6] Nicholson 
and Schuerger (2005) Astrobiology, 5, 536-544. [7] 
Schuerger et al (2003) Icarus, 165, 253-276. [8] 
Schuerger et al. (2006) Icarus, 181, 52-62. [9] Bell et 
al. (2000) JGR, 105, 1721-1755. [10] Gellert e tal. 
(2004) Science, 305, 829-832. [11] Reider et al. (2004) 
Science, 306, 1746-1749. [12] Boynton et al. (2004) 
LPSC 35th, Abst. #1950. [13] Schuerger et al. (2008) 
Icarus, 194, 86-100. [14] Mancinelli and Klovstad 
(2000) Planet. Sci., 48, 1093-1097. [15] Berry et al., 
(2009) Appl. Env. Microbiol. (in press).   


Biotoxicity of Mars Analog Soils: Microbial, Dispersal into Desiccated Soils Versus Emplacement in Salt or Ice Inclusions Fluids

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